Duolink In Situ - Fluorescence - Sigma
Contents
1.2. 4 1 4 Duolink In Situ Wash Buffers Duolink In Situ Wash Buffers contains 3 pouches of Wash Buffer A and one pouch of Wash Buffer B to be dissolved in high purity water 4 2 Reagents to be supplied by the user Reagents required for fixation and antigen retrieval of the sample according to your own protocol specific for each antigen and antibody used e Primary antibody or antibodies matching a set of PLA probes 4 3 Equipment needed e Fluorescence microscope equipped as follows Excitation emission filters compatible with fluorophore and nuclear stain excitation emission Camera and software for image acquisition optional e Staining jars e Pen or mask for delimitation of reaction area grease pen or silicon mask e g ImmEdge Pen from Vector Laboratories e Shaker Humidity chamber moist chamber Freeze block for enzymes Incubator 37 C Pipettes covering the range from 1 ul to 1000 ul Cover slips compatible with fluorescence microscopy e High purity water sterile filtered Milli Q or similar 4 4 Duolink ImageTool Software for Image Analysis The result from a Duolink In Situ experiment is typically a number of distinct fluorescent spots PLA signals of sub micrometer size in various locations of the studied cells Use Duolink ImageTool to obtain objective quantification of PLA signals The nuclei are automatically detected and cytoplasm size estimated enabling single cell statistica
3. Appendix C Duolink In Situ on cover slips References sssini 16 16 Ji 18 A ud 28 28 O o o nubs o Ww Un 1 Introduction Duolink In Situ reagents from Olink Bioscience enable detection visualization and quantification of individual proteins protein modifications and protein interactions in tissue and cell samples prepared for microscopy The target is detected using one or two primary antibodies depending on the application In the case that two primary antibodies are used they must have been raised in different species The Duolink In Situ reagents are based on in situ PLA which is a proximity ligation assay technology A pair of oligonucleotide labeled secondary antibodies PLA probes generates a signal only when the two PLA probes have bound in close proximity either to the same primary antibody or two primary antibodies that have bound to the sample in close proximity The signal from each detected pair of PLA probes is visualized as an individual fluorescent spot These PLA signals can be quantified counted and assigned to a specific subcellular location based on microscopy images 2 Principle of the assay Typical starting materials are adherent cells cytospin preparations or tissue sections on a glass slide fixed pre treated and blocked with a blocking reagent according to the requirements of the primary antibodies us
4. Ensure that no excessive amount of wash solution remains on slide before addition of ligation reagents Ensure that the Ligase is active i e has been kept at 20 C and that correct dilution of the reagents have been used Prepare fresh dilutions just before use do not allow mix with enzyme to stand for more than five minutes before use Inefficient amplification f the signals are very weak they can appear to be few as only a fraction reaches above detection threshold Ensure that no excessive amount of wash solution remains on the slide before addition of amplification reagents eep amplification time and temperature Ensure Polymerase is active i e has been kept at 20 C and that correct dilution of the reagents have been used Prepare fresh dilutions just before use do not allow mix with enzyme to stand for more than five minutes before use Problem High background general cause Probable cause Incomplete deparaffinization Suggested solution f you use paraffin embedded samples incomplete removal of paraffin can cause background Use fresh solutions if necessary and ensure that correct imes during deparaffinization have been used Custom blocking solution f you have used your own blocking solution allow primary antibody and PLA probes to be in contact with your blocking reagent before addition to he sample nsufficient washing of slides Ensure sufficient washing under gentle agitation a
5. 1 min d Let the slides dry at room temperature in the dark 7 Preparation for imaging Mount your slides with a cover slip using a minimal volume of Duolink In Situ Mounting Medium with DAPI ensuring no air bubbles get caught under the cover slip Nail polish can be used to seal the edges Wait for approximately 15 min before analyzing in a fluorescence or confocal microscope using at least a 20x objective After imaging store the slides at 20 C in the dark Duolink In Situ Mounting Media with DAPI is aqueous and does not solidify 8 1 Typical results The result from a Duolink In Situ experiment is typically a number of discrete fluorescent spots PLA signals in various locations of the studied cells see Figure 10 In some cases when studying highly expressed proteins the density of signals may be so high that the signals coalesce see Figure 11 A a Fig 10 Detection of EGFR in cytospin preparations of A431 cells using Duolink In Situ with one primary antibody where the primary antibody has been titrated to a very low concentration to give individual signals The pictures show a maximum intensity projection of the raw image based on 20 z planes PLA signals are shown in red and the nuclei in blue The nucleus image was acquired in one z plane A Positive reaction B Negative control without primary antibodies A B Fig 11 Detection of Her2 in FFPE preparations of SKBR 3 high expression cells using Duolink In Si
6. add 1ul of Ligase to 39 ul of Ligation solution Add the Ligation Ligase solution to each sample ncubate the slides in a pre heated humidity chamber for 30 min at 37 C Note Light sensitive reagents a Dilute the Amplification stock 1 5 in high purity water and mix ait to add the Polymerase until immediately before addition to the sample Take the addition of Polymerase into account when calculating the amount of water added Eg fora 40 ul reaction take 8 ul of the 5x Amplification stock and 31 5 ul of high purity water b Tapoffthe Ligation Ligase solution from the slides C Wash the slides in 1x Wash Buffer A for 2 x 2 min under gentle agitation Tap off all wash solution after the last washing d Remove the Polymerase from the freezer using a freezing block 20 C Add Polymerase to the Amplification solution from step a at a 1 80 dilution and vortex Eg fora 40 ul reaction add 0 5 ul of Polymerase to 39 5 ul of Amplification solution Add the Amplification Polymerase solution to each sample Incubate the slides in a pre heated humidity chamber for 100 min at 37 C Final wash step Note Light sensitive reagents Wash and dry the slides protected from light Ensure to have 1x and 0 01x Wash Buffer B available a Tapoffthe Amplification Polymerase solution from the slides b Wash the slides in 1x Wash Buffer B for 2 x 10 min c Wash the slides in 0 01x Wash Buffer B for
7. cause background C Add the primary antibody solution to each sample d Incubate in a humidity chamber Use temperature and time optimal for your primary antibodies 3 PLA probes Use the same buffer as for your primary antibodies or use Duolink In Situ Antibody Diluent Note The buffer should contain the blocking agent that was used for blocking the samples a Mixand dilute the two PLA probes 1 5 in your chosen buffer Allow the mixture to sit for 20 min at room emperature Eg for a 40 ul reaction take 8 ul of PLA probe MINUS stock 8 ul of PLA probe PLUS stock and 24 ul of the antibody buffer b Tapoffthe primary antibody solution from the slides c Washthe slides in a wash buffer suitable for your primary antibodies ashing should be performed in a staining jar with a minimum volume of 70 ml on a shaker cradle with gentle orbital shaking Bring the wash buffers to room temperature before use d Add the diluted PLA probe solution e ncubate the slides in a pre heated humidity chamber for 1 h at 37 C Proceed to the Detection protocol in section 7 3 on page 18 7 2 PLA probe protocol Duolink In Situ Solutions Follow this protocol if you use the Duolink In Situ Blocking Solution for blocking of your sample and Duolink In Situ Antibody Diluent for dilution of your primary antibodies Before you start your samples should be deposited on glass slides and pre treated with respect to fixatio
8. minimum volume of 70 ml is recommended Use fresh washing solutions and jars Drying of sample Ensure good humidity during all incubation steps and never let slides dry out after washes and before addition of reagents Unspecific Titrate primary antibodies IHC IF or PLA with respect to concentration binding of primary temperature time and buffer and ensure appropriate fixation protocol antibodies was used Large amounts of non Dust salt or fixation precipitates cause Discard wash solutions and use new solutions and washing jars If the problem remains sterile filter all washing solutions amplification highly fluorescent wash your cells at least twice to ensure that the culture medium is removed dependent particles before adding the fixative background Fluorescent Ink type pen has Never label slides with ink type pens Use diamond or graphite pen mist over been used entire slide Inappropriate wash Use Duolink In Situ Wash Buffers A and B during the Detection protocol buffers Inappropriate Use Duolink In Situ Mounting Medium with DAPI for mounting of the slides mounting medium Contamination in Discard wash solutions and use new solutions and washing jars wash solutions Red nappropriate wash Use Duolink In Situ Wash buffer B in the final washing step after the fluorescent buffers Amplification nuclei nappropriate mounting medium Use Duolink In Situ Mounting Medium with DAPI for mo
9. negative slide For proper positive and negative controls please refer to section 5 5 Controls on page 13 It is important to remember that a negative slide without primary antibodies but with PLA probes is likely to contain a few signals one or two signals every tenth cell and therefore the image from the negative reaction should not necessarily be completely blank The same setting should be used for all the images If the number of PLA signals is large it could happen that they coalesce all over the image or in certain cells Care must then be taken to choose for how to set the acquisition time whether to show the heavily expressed areas or to show all PLA signals even individual ones 8 3 Duolink ImageTool image analysis To analyze the results use Duolink ImageTool to obtain objective quantification of PLA signals By using the software it is possible to obtain either the number of signals and cells per image allowing average measurements or to allocate each individual signal to a specific cell using single cell analysis The nuclei are automatically detected and cytoplasm size estimated enabling single cell statistical analysis of expression levels in tissue or cell populations A result can typically look as shown in Figure 12 amp Duck Image Too 0 mages Manual luorescenca Fucrescence 1P EST Fie Help 2 4 06 imm H i 3 3 e Fig 12 Analysis of an image from a fluorescence microscope uc
10. on your primary antibodies Use the recommendations from the vendor of your primary antibodies if available If you are using chamber slides or cover slips follow the guidelines in Appendix B or C 5 4 1 Fixation Different fixations will influence the antigenicity and thus the performance of your primary antibodies If there is no fixation recommendation for your antibody you must optimize this for your application For cell materials like cells grown on slides or cytospin preparations you should choose the fixation most suitable for your primary antibody The best choice of fixation may vary depending on your cell material antibody and the type of protein you want to study Duolink In Situ is compatible with all fixations typically used for IHC including e Ethanol e Zinc Acetone Formalin Paraformaldehyde PFA 12 5 4 2 Antigen Epitope Retrieval Antigen epitope retrieval is mostly used for formalin fixed paraffin embedded FFPE material Formalin fixation is masking the epitopes which then can be un masked by antigen epitope retrieval Different methods for antigen epitope retrieval will influence the performance of your primary antibodies and must be optimized for your application Duolink In Situ is compatible with all antigen retrieval methods typically used for IHC including Heat Induced Epitope Retrieval HIER e Enzyme treatment with Proteinase K Pepsin Trypsin etc For HIER it might be necessary to try a few dif
11. shaker during the washing steps and add as much wash buffer as possible Use the same washing times as recommended in the Duolink In Situ assay protocol Make sure that the cells do not dry out when changing wash buffer Mounting Mount each cover slip by turning it upside down on a regular microscope slide using a minimal volume of Duolink In Situ Mounting Medium with DAPI in between More details are found in section 7 3 chapter 7 in the Duolink In Situ User Manual References Jarvius M Paulsson J Weibrecht Leuchowius KJ Andersson AC W hlby C Gullberg M Botling J Sj blom T Markova B Ostman A Landegren U S derberg O In situ detection of phosphorylated platelet derived growth factor receptor B using a generalized proximity ligation method Molecular and Cellular Proteomics 6 1500 1509 2007 S derberg O Gullberg M Jarvius M Ridderstr le K Leuchowius KJ Jarvius J Wester K Hydbring P Bahram F Larsson LG and Landegren U Direct observation of individual endogenous protein complexes in situ by proximity ligation Nat Methods 3 995 1000 2006 Gullberg M Gustafsdottir SM Schallmeiner E Jarvius J Bjarneg rd M Betsholtz C Landegren U and Fredriksson S Cytokine detection by antibody based proximity ligation Proc Natl Acad Sci USA 101 8420 24 2004 Fredriksson S Gullberg M Jarvius J Olsson C Pietras K Gustafsdottir SM Ostman A and Landegren U Protein detection using proximity dependent DNA l
12. system Alternatively if you are using an approach with two primary antibodies you can also choose to omit only one of your primary antibodies If facing problems with background signals with the primary antibodies omitted consult the troubleshooting guide see section 9 6 Reagent preparation 6 1 Duolink In Situ reagents Some Duolink In Situ reagents are supplied as concentrated stocks Dilute required volumes of the stocks Note Do not store diluted reagents Blocking Solution store at 4 C Vortex before use e Ready to use blocking solution one drop equals approximately 40 ul Antibody Diluent store at 4 C Vortex before use For dilution of primary antibodies and PLA probes PLA probe 5x store at 4 C Vortex before use Dilute 1 5 in Antibody Diluent or custom diluent immediately before use and vortex the solution before addition to the sample Ligation 5x store at 20 C Thaw at room temperature and vortex before use Dilute 1 5 in high purity water immediately before use Note The buffer contains DTT that may precipitate during freezing Vortex to dissolve homogenize Ligase 1 U ul store at 20 C e The Ligase should be kept at 20 C at all times Use a freezing block 20 C when removing the enzyme from the freezer Add the Ligase to the reaction mix at a 1 40 dilution immediately before addition to the sample Ensure that the Ligation Ligase reaction solution is thoro
13. User Guide Duolink In Situ Fluorescence sigma com SIGMA ALDRICH The protocols in this manual are compatible with all Duolink In Situ PLA probes Duolink In Situ Detection Reagents Green Cat No DUO92014 Orange Cat No DUO92007 Red Cat No DUO92008 and Far Red Cat No DUO92013 Duolink In Situ Fluorescence Table of Contents da JREOQUGHOD eate ett beste mun call 2 Principle of the assay ttn 3 Applications 31 Detect and quantify protein interactions 32 Detect and quantify protein modifications 33 Detect and quantify protein expression 4 Reagents and equipment 4 4 Duolink In Situ reagents 4 2 Reagents to be supplied by the user 43 Equipment needed 44 DuolinkImageTool software for image analysis 5 Assay considerations 5 1 Choice of primary antibodies 52 Primary antibody optimization 5 3 Sample type 54 Pre treatment 5 5 COMMUN Sp asini NR mei DN 6 Reagent preparation 6 11 DuolinkIn Situ reagents 6 2 Duolink In Situ Wash Buffers 6 33 Samples 64 Reaction volume tt 7 Assay protocol 7 PLA probe protocol CUSTOM solutions 7 2 PLA probe protocol Duolink In Situ Solutions 73 Detection protocol 8 Results senes 8 1 Typical results 8 2 Image acquisition 83 Duolink ImageTool image analysis 9 Troubleshooting Appendix A Wash Buffers formulas Appendix B Duolink In Situ in chamber slides
14. a fixative of your choice e g freshly thawed 3 PFA in 1xPBS Fix the slides according to your protocol preferably with gentle agitation 32 Rinse three times with wash buffer e g cold 1xPBS 2 min per rinse for PFA fixation add 200 ul 1M glycine Coplin jar in the second wash 3 Wash quickly in MilliO water once to remove salt 4 Usethe slides directly in your Duolink In Situ assay or dry and store the slides at 20 C When drying the slides try to speed up the drying process before freezing in order to avoid degradation of proteins at room temperature 5 Delimit the reaction area by using a grease pen before proceeding with the Duolink In Situ assay protocol 25 26 Appendix C Duolink In Situ on cover slips Place the cover slips in a multiwell plate when running the Duolink In Situ assay Mark the edges of the cover slip with a hydrophobic pen to delimit the reaction area Use the Reaction Volume Guide at sigma com duolink for guidelines on how much reaction volume is needed per reaction area Incubation steps Make sure that the cells do not dry out and that the temperature is kept at 37 C during the incubation steps In order to improve heat transfer and ensure an even temperature throughout the plate use the Duolink In Situ Microplate Heat Transfer Block Cat No DUO82065 pre heated to 37 C Wash steps Perform the washing steps with the cover slips still in the multiwell plate Put the plate on a
15. ary antibody with another blocking solution Supplied in ready to use concentration e Antibody Diluent For dilution of PLA probes and in some cases the primary antibodies section 5 4 3 Supplied in ready to use concentration PLA probe 5x stock Secondary antibody conjugated with a PLA oligonucleotide 4 1 2 Duolink In Situ Detection reagents Duolink In Situ Detection Reagents are available with four different fluorophores Detection Reagents Excitation nm Emission nm Filters for image acquisition Detection Reagents Green 495 527 same filters as e g Cy2 or FITC Detection Reagents Orange 554 579 same filters as e g Cy3 Detection Reagents Red 594 624 same filters as e g Texas Red Detection Reagents Far Red 644 669 same filters as e g Cy5 Each Duolink In Situ Detection Reagents box contains the following Ligation 5x Contains oligonucleotides that hybridize to the PLA probes and all components needed for ligation except the Ligase Ligase 1 U ul e Amplification 5x Contains all components needed for Rolling Circle Amplification except the Polymerase Included are also oligonucleotide probes labeled with a fluorophore that hybridize to the RCA product Polymerase 10 U ul 4 1 3 Duolink In Situ Mounting Medium with DAPI Optimal mounting medium for preservation of the PLA signals It also contains DAPI nuclear stain excitation 360 nm and emission 460 nm Aqueous mountant which does not solidify
16. at room temperature Expiry date is marked on each individual lot 1x solutions may be kept at room temperature for short time storage one week or less For long time storage store at 4 C Bring the solutions to room temperature before use Alternative 2 make your own Duolink In Situ Wash Buffer A Dissolve 8 8 g NaCl 1 2 g Tris base and 0 5 ml Tween 20 in 800 ml high purity water Adjust pH to 7 4 using HCI Add high purity water to 1000 ml final concentrations 0 01 M Tris 0 15 M NaCl and 0 0596 Tween 20 Filter the solution through a 0 22 um filter and store at 4 C Bring the solutions to room temperature before use Duolink In Situ Wash Buffer B Alternative 1 use Duolink In Situ Wash Buffer B Cat No DUO82048 To prepare a 1x buffer dissolve the content of one pouch in high purity water to a final volume of 1000 ml Store pouches at room temperature Expiry date is marked on each individual lot 1x solutions may be kept at room temperature for short time storage one week or less For long time storage store at 4 C Bring the solutions to room temperature before use Alternative 2 make your own Duolink In Situ Wash Buffer B Dissolve 5 84 g NaCl 4 24 g Tris base and 26 0 g Tris HCl in 500 ml high purity water Adjust pH to 7 5 using HCI Add high purity water to 1000 ml final concentrations 0 2 M Tris and 0 1 M NaCl Filter the solution through a 0 22 um filter and store at 4 C Bring the solutions to room temperat
17. e time concentration and buffer composition Insufficient reaction volume Ensure that your reaction area corresponds to the reaction volume The droplet must cover the reaction area Encircle your reaction area using a hydrophobic barrier and incubate the slides in a humidity chamber to prevent evaporation Do NOT use a cover slip to disperse the droplet Mounting media The intensity of the PLA signals will decrease quickly in some mounting media Duolink In Situ Mounting Medium with DAPI is optimized for preserving the PLA signals Wrong filter used for acquisition The fluorophore in the Amplification Green has an excitation wavelength of 495 nm and an emission wavelength of 527 nm and can be detected using he same filters as for e g Cy2 or FITC The fluorophore in the Amplification Orange has an excitation wavelength of 554 nm and an emission wavelength of 579 nm and can be detected using he same filters as for e g Cy3 The fluorophore in the Amplification Red has an excitation wavelength of 594 nm and an emission wavelength of 624 nm and can be detected using the same filters as for e g Texas Red The fluorophore in the Amplification Far Red has an excitation wavelength of 644 nm and an emission wavelength of 669 nm and can be detected using he same filters as for e g Cy5 Make sure that you use appropriate filters Inefficient ligation eep ligation incubation time and temperature
18. e at a time using Duolink In Situ single recognition or possibly IF or IHC Use proper controls to verify that your individual antibodies do bind the correct target under the conditions used 5 5 Controls To be able to properly evaluate your results it is advisable to include both biological and technical controls Examples of suitable controls are given below 5 5 1 Positive control Ideally you should include a cell or tissue type that you know contains your target protein and that your primary antibodies should bind to If you are using two primary antibodies the primary antibodies should be known to bind in close proximity This also enables you to verify the Duolink In Situ procedure 5 5 2 Negative control Ideally you should include a negative control with a cell line or tissue that does not express one or both of your targets Such a control will give you information on the specificity of your primary antibodies If this control gives you background signals in excess of signal obtained from omitting the primary antibody antibodies it means that your primary antibodies under the present conditions are binding to other targets than expected If facing problems with signals in your negative cell line tissue you might need to titrate your primary antibodies or look for more specific antibodies 5 5 3 Negative control technical By omitting primary antibodies you will get a hint of how the PLA probe background looks like in your
19. ed A The samples are incubated with primary antibodies that bind to the protein s to be detected B Secondary antibodies conjugated with oligonucleotides PLA probe MINUS and PLA probe PLUS are added to the reaction and incubated C TheLigation solution consisting of two oligonucleotides illustrated as red bands and Ligase is added and the oligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity D The Amplification solution consisting of nucleotides not shown and fluorescently labeled oligonucleotides is added together with Polymerase The oligonucleotide arm of one of the PLA probes acts as a primer for a rolling circle amplification RCA reaction using the ligated circle as a template generating a concatemeric repeated sequence product The fluorescently labeled oligonucleotides will hybridize to the RCA product The signal is easily visible as a distinct fluorescent spot and analyzed by fluorescence microscopy Fig 1 Assay principle 3 Applications 3 1 Detect and quantify protein interactions Detection and quantification of interacting proteins can be done using two different primary antibodies against each of the two proteins of interest 3 2 Detect and quantify protein modifications Detection and quantification of a protein and its specific post translational modification can be done using two different primary antibodi
20. es one directed against the target protein and one against a modification site on the same protein 3 3 Detect and quantify protein expression Detection and quantification of protein expression can be done with two different approaches A To detect and quantify one single protein with high sensitivity use only one primary antibody B To detect and quantify one single protein with high specificity use two different primary antibodies directed against two different epitopes on the same protein Fig 2 Protein interactions Fig 3 Protein modifications P P Fig 4 A Single recognition Fig 5 B Double recognition 4 Reagents and equipment 4 1 Duolink In Situ reagents The Duolink In Situ reagents are generic reagents using secondary antibodies for detecting presence of analyte specific primary antibodies provided by the user To run a Duolink In Situ assay you need the following Duolink In Situ components PLA probe MINUS e Wash Buffers A and B PLA probe PLUS e Mounting Medium with DAPI e Detection Reagents 4 1 1 Duolink In Situ PLA probes The choice of PLA probes depends on the species of your primary antibodies and your application see section 3 For a complete list of PLA probes visit sigma com duolink Each Duolink In Situ PLA probe product contains the following Blocking Solution For blocking of the sample if you have not already optimized your prim
21. ferent solutions of various pH 5 4 3 Blocking solution and Antibody diluent It is essential to use a proper blocking solution and antibody diluent Use the recommendations from the vendor of your primary antibodies if available If you have previously optimized your assay in for example IHC use the same conditions for Duolink In Situ There are two different protocols depending on whether you use your own blocking solution and antibody diluent or the Duolink In Situ Blocking Solution and Antibody Diluent PLA probe protocol CUSTOM solutions e Use this protocol if you use your own blocking and antibody diluent recommended see section 7 1 The same agent used for blocking of the sample should be present also in the antibody diluent preferably together with some detergent to reduce background staining as well as in the diluent for the PLA probes Note Ensure that you do not use bulk IgG from the same species as your primary antibodies as blocking reagent as this will cause false signals from the PLA probes PLA probe protocol Duolink In Situ Solutions Use this protocol if you have no previous experience or no recommendation from the antibody vendor regarding blocking and antibody diluents see section 7 2 Note The Duolink In Situ Antibody Diluent contains salt blocking agent and detergents All to prevent unspecific binding of the antibodies It is important that you first verify the function of your primary antibodies on
22. igation assays Nat Biotechnol 20 473 77 2002 27 All third party trademarks are the property of their respective owners This product is covered by several patents and patent applications including US 6 511 809 US 6 558 928 US 6 8785 15 US 7 074 564 US 5 665 539 and related US and foreign patents This product is for research use only Not for use in human diagnostic or therapeutic procedures This product includes a license for non commercial use of the Duolink product Commercial users will require additional licenses Please contact Olink AB for details There are no warranties expressed or implied which extend beyond this description Olink AB is not liable for property damage personal injury or economic loss caused by this product Order Customer Service 800 325 3010 Fax 800 325 5052 World Headquarters Technical Service 800 325 5832 sigma aldrich com techservice 3050 Spruce St Development Custom Manufacturing Inquiries SAFC 800 244 1173 St Louis MO 63103 e 314 771 5765 Safety related Information sigma aldrich com safetycenter sigma aldrich com O 2013 Sigma Aldrich Co LLC All rights reserved SAFC SIGMA and SIGMA ALDRICH are trademarks of Sigma Aldrich Co LLC registered in the US and other countries Where bio begins is a trademark of Sigma Aldrich Co LLC Content and images are O 2012 Olink AB and used with permission Duolink and PLA are trademarks of Olink AB SIGMA ALDRICH 80021 1033
23. l analysis of expression levels in tissue or cell populations Furthermore regions of interest can be defined a feature of particular relevance when studying tissue samples Raw imaging data can be imported directly from the four major microscope vendors Olympus Leica Nikon and Zeiss The results data can be exported into an Excel sheet for further evaluation 5 Assay considerations 5 1 Choice of primary antibodies The Duolink In Situ reagents are generic reagents using secondary antibodies for detecting presence of analyte specific primary antibodies Your choice of primary antibodies is crucial when setting up the PLA assay The primary antibodies should be of IgG class specific for the target to be detected and preferably affinity purified The primary antibodies could be either polyclonal or monoclonal To maximize your success rate choose antibodies that are IHC and or IF classified and follow the optimization guidelines given in section 5 2 Primary antibodies could also be qualified individually in a Duolink In Situ single recognition experiment section 5 1 1 before a double recognition assay 5 1 1 Primary antibodies for detection of protein expression When single protein targets are to be detected you can use either one or two primary antibodies against your target A One primary antibody single recognition Usage of one primary antibody gives you an easy to set up assay with high J F sensitivity In this case o
24. lei are blue the red spots are the PLA signals representing the protein target of interest PLA signals marked with white circles and nuclei outlined in yellow are quantified at analysis The green outlines represent the user defined cytoplasm size When studying highly expressed proteins the density of PLA signals may be so high that it is impossible to discern the individual signals see Figure 11 Duolink ImageTool can not quantify regions of merged signals only separable signals can be counted For images taken with fluorescence microscopy a quality control function displays a warning if signals are merged In this case the data is best analyzed by fluorescence intensity measurement or area fraction of sample with signal using software for traditional fluorescence analysis The primary antibodies used in the assay can be titered down to reduce the number and incidence of merged signals 21 22 9 Troubleshooting Some general guidelines are given below Problem Probable cause No or few No or insufficient signals in binding of primary positive antibodies samples Suggested solution Optimize your primary antibodies individually with the Duolink In Situ single recognition approach IHC or IF in positive control samples Optimize parameters such as fixation retrieval protocol blocking and buffer conditions etc for the subsequent Duolink In Situ double recognition assay Evaluate and optimize fixative retrieval incubation temperatur
25. n retrieval and or permeabilization If you are using chamber slides or cover slips follow the guidelines in Appendix B or C Use open droplet reactions without a cover slip and perform all incubations in a humidity chamber The volume examples are based on 40 ul reaction volume suitable for 1 cm Use volumes corresponding to your delimited reaction area see the Reaction Volume Guide at sigma com duolink or section 6 4 1 Blocking a Add one drop of Blocking Solution per 1 cm Ensure to cover the entire reaction area with Blocking Solution b Incubate the slides in a pre heated humidity chamber for 30 min at 37 C 2 Primary Antibodies a Dilute your primary antibody or antibodies to suitable concentration in the Antibody Diluent b Tapoffthe Blocking Solution from the slides Try to obtain an equal residual volume on each slide as this will affect reproducibility Do not allow the samples to dry before adding the primary antibodies as this will cause background c Add the primary antibody solution to each sample d Incubate in a humidity chamber Use the optimal incubation temperature and time for your primary antibodies 3 PLA probes a Dilute the two PLA probes 1 5 in Antibody Diluent Eg fora 40 ul reaction take 8 ul of PLA probe MINUS stock 8 ul of PLA probe PLUS stock and 24 ul of Antibody Diluent b Tapoffthe primary antibody solution from the slides c Washthe slides in 1x Wash Buffer A or i
26. n a wash buffer suitable for your primary antibody preferably 2 x 5 min Washing should be performed in a staining jar with a minimum volume of 70 ml on a shaker cradle with gentle orbital shaking Bring the wash buffers to room temperature before use d Add the PLA probe solution e Incubate the slides in a pre heated humidity chamber for 1 h at 37 C Proceed to the Detection protocol in section 7 3 on page 18 7 3 Detection protocol Proceed from step 3e of the PLA probe protocols Use open droplet reactions without a cover slip and perform all incubations in a humidity chamber The volume examples are based on 40 ul reaction volume suitable for 1 cm reaction area Adjust the volumes corresponding to your specific delimited reaction area see the Reaction Volume Guide at sigma com duolink or section 6 4 a Dilute the Ligation stock 1 5 in high purity water and mix Wait to add the Ligase until immediately before addition to the samples Take the addition of Ligase into account when calculating the amount of water added Eg fora 40 ul reaction take 8 ul of the 5x Ligation stock and 31 ul of high purity water b Tapoffthe PLA probe solution from the slides C Wash the slides in 1x Wash Buffer A for 2 x 5 min under gentle agitation d Remove the Ligase from the freezer using a freezing block 20 C Add Ligase to the Ligation solution from step a at a 7 40 dilution and vortex Eg for a 40 ul reaction
27. nly one antibody needs to bind the target under the e conditions used fixation retrieval buffer etc Using one primary antibody is recommended for detection of single target proteins only when a well performing specific primary antibody is available If you use an unspecific antibody you will see high background due to the sensitivity of the assay Using only one primary antibody is more efficient than using two primary antibodies when detecting low abundant targets Fig 6 A Single recognition B Two primary antibodies double recognition Usage of two primary antibodies will give you an assay with superior specificity Fi When using two primary antibodies they must be directed against different non competing epitopes on the same target molecule The two primary antibodies must have been raised in different species Also both primary antibodies must bind to the target under the same conditions fixation retrieval buffer etc Fig 7 B Double recognition 5 1 2 Primary antibodies for detection of protein modifications F Detection of protein modifications such as phosphorylations often suffers from low specificity Our recommended strategy is to use two primary antibodies one against the target protein and one against a modification site on the same protein Preferably a specific modification site is targeted but it is also possible to use a generic antibody against a modification site Using two primary a
28. ntibodies will give you superior specificity for your modification assay compared to using one single primary antibody The two primary antibodies must be raised in different species Also both primary antibodies must bind to the target under the same conditions fixation retrieval buffer etc Fig 8 Protein modifications 5 1 3 Primary antibodies for detection of protein interactions Duolink In Situ provides an excellent way to detect protein interactions This is done using two primary antibodies each directed against one of the targets of interest The two primary antibodies must be raised in different species and must bind to the target under the same conditions fixation retrieval buffer etc Fig 9 Protein interactions 5 2 Primary antibody optimization You will save time and increase your success rate if you optimize your primary antibodies to be sure that they work properly in your material The conditions for your primary antibodies should be optimized with respect to Sample fixation Antibody diluent Antigen retrieval e Primary antibody concentration Blocking solution Sample pre treatment for Duolink In Situ is identical to procedures used for immunohistochemical IHC and or immunofluorescence IF staining If you already have a working assay for IHC or IF use the same pre treatment protocol The primary antibody concentration may need to be titrated and optimized for the Duolink In Si
29. re To prepare a 1x buffer dissolve the content of one pouch in high purity water to a final volume of 1000 ml Store pouches at room temperature 1x solutions may be kept at room temperature for short time storage one week or less For long time storage store at 4 C Bring the solutions to room temperature before use e Forthe final wash step prepare 0 01x Wash Buffer B by diluting 1x buffer 1 100 in high purity water e Alternatively make your own Duolink In Situ Wash Buffer B according to Appendix A 6 3 Samples Before you start the Duolink In Situ protocol the sample should have been deposited on a glass slide sufficiently pre treated to fit your primary antibodies with respect to fixation retrieval and permeabilization see section 5 2 Your reaction area must be delimited with e g a grease pen or silicon mask If you are using chamber slides or cover slips follow the guidelines in Appendix B or C 6 4 Reaction volume Use open droplet reactions 02 cm 15 ul Tem 40 ul e Perform the incubations without a cover slip E 2 cm 80 ul Perform all incubations in a pre heated M 3cm 120 ul humidity chamber 4cm 160 ul Use volumes corresponding to your delimited E 6cm 240 ul reaction area Never use less than 15 ul of total reaction volume see Table 1 or the Reaction 8cm 320 ul Volume Guide at sigma com duolink 10 cm 400 ul Note It is important that all incubations are perfo
30. rmed in a humid environment to prevent excessive evaporation If the sample goes dry this will give rise to severe artifacts Table 1 Suitable reaction volume for different reaction areas 7 Assay protocol 7 1 PLA probe protocol CUSTOM solutions Follow this protocol if you use your own blocking solution and antibody diluent that you know work with your primary antibodies Before you start your samples should be deposited on glass slides and pre treated with respect to fixation retrieval and or permeabilization If you are using chamber slides or cover slips follow the guidelines in Appendix B or C Use open droplet reactions without a cover slip and perform all incubations in a humidity chamber Use volumes corresponding to your delimited reaction area see the Reaction Volume Guide at sigma com duolink or section 6 4 1 Blocking Use your previously tested blocking solution v Add blocking solution to each sample o Incubate the slides Primary Antibodies Use your previously tested buffer to dilute your primary antibodies a Dilute your primary antibody to a suitable concentration in your custom antibody diluent If using two primary antibodies dilute the two antibodies in the same diluent b Tap off the blocking solution from the slides Try to obtain an equal residual volume on each slide as this will affect reproducibility Do not allow the samples to dry before adding the primary antibodies as this will
31. tu conditions When using two primary antibodies optimization is best done for one primary antibody at a time using the Duolink In Situ single recognition approach These conditions can then be used as a start for optimizing your assays using both primary antibodies in the same Duolink In Situ assay When using two primary antibodies it may in some cases be difficult to find conditions compatible with both of the selected primary antibodies Under these circumstances it is advisable to search for other antibodies recognizing alternative epitopes For further advice on immunostaining optimization we recommend the Education Guide from Dako http www dako com 08002 ihc staining methods 5ed pdf 5 3 Sample type Duolink In Situ reagents can be used with a broad range of sample types depending on the preferences of your primary antibodies Duolink In Situ has so far shown to be successful with the following sample types Adherent cell cultures Formalin fixed paraffin embedded tissue e Cytospin preparations SECUONS e Formalin fixed paraffin embedded cell samples Frozen tissue sections e Fresh tissue sections 5 4 Pre treatment Before you start your samples should be deposited on glass slides and sufficiently pre treated with respect to fixation retrieval and permeabilization It is crucial for the performance of the assay to optimize the conditions for the primary antibodies Also the choice of wash buffer may be dependent
32. tu with two primary antibodies In this case the antibodies have been titrated to give signals in low expression cell lines The pictures show a maximum intensity projection of the raw image based on 20 z planes PLA signals are shown in red and the nuclei in blue The nucleus image was acquired in one z plane A Positive reaction B Negative control without primary antibodies 20 8 2 Image acquisition The PLA signal is recognized as a fluorescent spot see Figure 10 in a fluorescence microscope using the appropriate filters for the detection fluorophore used An individual signal is of sub micrometer size For images taken in one focal plane several signals can be either above or below the current focus A true PLA signal is easy to go through by changing the focus making it appear and disappear This does not apply to coalesced signals which may occur for highly expressed proteins see below To detect all PLA signals it is thus necessary to obtain images throughout the entire thickness of the sample However you may acquire images in one plane as long as all images to be compared are acquired in a similar position within the sample As with all fluorescent imaging it is important to keep settings constant during an experiment with regard to exposure time and filters used etc It is recommended to get a feeling for the setting by use of one acquisition time needed for your specific microscope setting by use of one positive slide and one
33. ughly vortexed before addition to the sample Amplification 5x store at 20 C Thaw at room temperature and vortex before use Dilute 1 5 in high purity water immediately before use Polymerase 10 U ul store at 20 C The Polymerase should be kept at 20 C at all times Use a freezing block when removing the enzyme from the freezer Add the Polymerase to the reaction mix at a 1 80 dilution immediately before addition to the sample Ensure that the Amplification Polymerase reaction solution is thoroughly vortexed before addition to the sample 6 2 Duolink In Situ Wash Buffers The washing steps should be performed in a staining jar with a minimum volume of 70 ml on a shaker cradle with gentle orbital shaking Washing after the primary antibodies should be performed in Duolink In Situ Wash Buffer A or in the wash buffer optimal for your primary antibodies In all subsequent wash steps ensure to use Duolink In Situ Wash Buffers Wash Buffer A store at room temperature e To prepare a 1x buffer dissolve the content of one pouch in high purity water to a final volume of 1000 ml e Store pouches at room temperature 1x solutions may be kept at room temperature for short time storage one week or less For long time storage store at 4 C Bring the solutions to room temperature before use e Alternatively make your own Duolink In Situ Wash Buffer A according to Appendix A Wash Buffer B store at room temperatu
34. unting of the slides Large variation of signals over slide individual reaction Uneven spread of reagents during different steps Ensure that entire area of investigation is covered during each step Partial drying of parts of the area during incubation Check before each new step that no region has dried during incubation f so use a better humidity chamber The reaction spreads over atoo large surface is unable to cover sample No or insufficient delimitation around reaction area Use a grease pen or alike to encircle reaction area Inappropriate reaction volume Adjust reaction volume according to your reaction area For guidance on reaction volumes please refer to section Reagent Preparation or the separate Reaction Volume Guide No nuclear staining is observed Mounting without mounting media containing DAPI Ensure using Duolink In Situ Mounting Medium with DAPI Wrong filter used for acquisition Nuclear staining is performed with DAPI excitation 360 nm and emission 460 nm Correct filters must be used If problems remain contact us at sigma aldrich com techservice or 800 325 5832 23 24 Appendix A Wash Buffers formulas Duolink In Situ Wash Buffer A Alternative 1 use Duolink In Situ Wash Buffer A Cat No DUO82047 To prepare a 1x buffer dissolve the content of one pouch in high purity water to a final volume of 1000 ml Store pouches
35. ure before use Appendix B Duolink In Situ in chamber slides The following protocol describes how to wash and fix cells that are placed on chamber slides before using them in a Duolink In Situ assay In order to avoid drying of the samples prepare just a few slides at a time Culture and treat your cells in a container of your choice e g flasks or chamber slides Before fixation cells should be plated in chamber slides and allowed to grow to desired confluency Fixation should be done as soon as possible after harvesting the cells in order to minimize degradation of proteins If cells are grown too dense it may be difficult to separate nuclei clusters in the final image analysis Washing before fixation 1 Pour out the cell culture medium by quickly inverting the chamber slides Quickly add cold wash buffer of your choice e g 1xPBS so the wells are completely filled to prevent the cells from drying 2 First wash Take one slide at a time and pour out the wash buffer by inverting the slide Fill up immediately with fresh wash buffer 3 Second wash Repeat step 2 4 Third wash Remove the chambers while the wash buffer remains in the wells Do not let the cells dry out If your slide contains a silicon barrier remove it as well Quickly place the slide in a Coplin jar with cold wash buffer and shake it manually for a few seconds Fixation 1 Transferthe slides from the Coplin jar with wash buffer to a new Coplin jar with
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