PALM MicroLaser Systems
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1. 100x magnifying objectives 0 17 mm thin cover glass slides have to be used e Samples on glass slides With the PALM MicroBeam almost every kind of biological material can be microdissected and catapulted directly from glass slides Even archival pathological sections can be used after removing the cover slip and the mounting medium To facilitate easy catapulting additional adhesive substances or Superfrost charged slides should only be applied when absolutely necessary for the adhesion of special material e g brain sections e Samples on membrane slides PALM MembraneSlides MembraneSlides are special slides covered with a membrane The membrane is easily cut together with the sample and acts like a stabilizing backbone during catapulting Therefore even large areas are catapulted by a single laser pulse without affecting the morphological integrity The membrane facilitates catapulting of even large areas with one single laser pulse This feature is especially important for isolated single cells chromosomes and also living cells or small organisms P A L M offers slides with PEN membrane This polyethylene naphthalate membrane is 1 35 um thick and is highly absorptive in the UV A range which facilitates laser cutting The PEN membrane can be used for all applications There are also MembraneSlides available covered with POL polyester membrane which is 0 9 um thick and less sensitive to the UV laser e g a higher laser2. RNA preparations are planned with these frozen sections we at PALM recommend ethanol fixation This is performed after 20 seconds of air drying on ice by dipping the mounted sections for 1 5 minutes into icecold 20 C 70 ethanol Removing the freeze supporting substance If OCT or another tissue freezing medium is used it is important to get rid of the media on the slide before Laser Microdissection because these media will interfere with laser efficiency Removing of the medium is very easily done by gently washing the slide for about 1 minute in water If the sections are stained the supporting substance is removed automatically in the aqueous staining solutions or the diluted ethanols Frozen sections should always be allowed to dry for 5 up to 30 minutes at room temperature before use e Cytospins Cytospins can be prepared on glass slides or on MembraneSlides After centrifugation with a cytocentrifuge let the cells air dry over night Then fix for 5 minutes in 100 methanol Allow the cytospins to dry at room temperature before staining Blood and tissue smear Distribute a drop of peripheral blood or material of a swab smear over the slide Let smears shortly air dry and fix them for 2 up to 5 minutes in 70 ethanol gt Staining of the sections Using frozen tissue one has to be aware of endogeneous RNases that may still be active after short fixation steps Therefore it is recommended to keep all incu
3. be kept on ice e Store RNA aliquoted in ethanol or RNA buffer at 80 C Most RNA is relatively stable at this temperature Store prepared slides also at 80 C e RNA is not stable at elevated temperatures therefore avoid high temperatures gt 65 C since these affect the integrity of RNA DEPC treatment of water and solutions see at page 6 gt Preparation of frozen sections Prepare your sections onto the slide as you do routinely Let 20 seconds air dry on ice Fix in 70 ice cold ethanol for 1 5 minutes dip in RNase free water perform your preferred staining e g hematoxylin dip into 70 96 and 100 ethanol respectively Subsequently let air dry for 5 15 minutes The slides can now be used at once even if they are somewhat wet or deep frozen at 80 C Procedure For cryosections we perform catapulting of the cells into a buffer without Proteinase K If using paraffin sections for microdissection please use a buffer containing Proteinase K like mentioned in the DNA protocol also see below Best use freshly cut and stained specimens Protocols_EN_0403 doc 11 14 PALM MicroLaser Systems d PA L M Protocols Microlaser Technologies gt Preparation of RNA from catapulted samples To ensure RNase free membrane slides we at P A L M dip them for a few seconds into RNase ZAP Ambion 9780 250 ml followed by two separate washings in DEPC treated water and drying at 37 up to 5
4. with a heating lid for the standard digestion If not going on immediately store the samples in the fridge at 4 C ua A UNBE Oo C9 The time necessary for complete digestion depends on the kind and on the number of catapulted cells Protocols_EN_0403 doc 10 14 PALM MicroLaser Systems d PA L M Protocols Microlaser Technologies gt Some special tips for working with RNA Working with RNA is more demanding than working with DNA because of the chemical instability of the RNA and the ubiquitous presence of RNAses e It makes sense to designate a special area for RNA work only e Clean benches with 100 ethanol or a special cleaning solution e g RNaseZap Ambion 9780 or similar e Always wear gloves After putting on gloves do not touch surfaces or equipment to avoid reintroduction of RNases to decontaminated material e Use sterile disposable plasticware e Use filtered pipetter tips e Glassware should be baked at 180 C for 4 hours RNases can maintain activity even after prolonged boiling or autoclaving e Purchase reagents that are RNAse free e All solutions should be made with DEPC diethylpyrocarbonate treated H20 e Treat all used material with DEPC e For best results use either fresh samples or samples that have been quickly frozen in liquid nitrogen or at 80 C This procedure minimizes degradation of RNA by limiting the activity of endogeneous RNAses All required reagents should
5. 3 minutes rinsing in RNase free tap water or blueing solution 30 seconds up to 3 minutes Eosin Y e g SIGMA HT110 2 32 quick increasing ethanol series let air dry at room temperature o Methylene Blue The nuclei are stained dark blue Not recommended for RNA extraction Procedure directly from distilled water 5 10 minutes Methylene Blue solution 0 05 in water SIGMA 31911 2 rinsing in Aqua dest let air dry at room temperature o Methyl Green The nuclei are stained dark green the cytoplasm light green Procedure directly from distilled water 5 minutes Methyl Green solution DAKO S1962 rinsing in Aqua dest let air dry at room temperature o Nuclear Fast Red The nuclei are stained dark red the cytoplasm lighter red Procedure directly from distilled water 5 to 10 minutes Nuclear fast red solution DAKO S1963 rinse in A dest let air dry at room temperature Protocols_EN_0403 doc 7 14 PALM MicroLaser Systems d PA L M Protocols Microlaser Technologies gt Laser cutting microdissection of the samples Please have a look also in the PALM MicroBeam User Manual e Tricks for improvement of the morphology sight of sections o Ethanol Go forward to search an interesting area on the section and pipet about 5 ul of ethanol onto this area You may use 70 or 100 ethanol When using absolute ethanol a little destaining of the section may happen but the drying is much quic
6. 5 C for 30 minutes up to 2 hours Afterwards the standard UV treatment see page 4 is performed as usual shortly before use To reduce the chance of contamination with exogenous RNases we use only special reagents and solutions for RNA isolation reverse transcription and RT PCR All used solutions and tubes are prepared with DEPC treated water or are purchased as guaranteed RNase free We also recommend the use of filter tips Best results are obtained using freshly prepared cryosections Catapult Buffer 0 5 M EDTA pH 8 0 20 ul 1M Tris pH 8 0 200 ul Igepal CA 630 SIGMA I 3021 50 ul optional Proteinase K 20 mg ml 100 ul ddH20 DEPC treated autoclaved fill up to 10 ml Proteinase K solution 20 mg ml Qiagen GmbH Hilden Germany Catalog 19131 only necessary for paraffin sections Always prepare a fresh mixture of Proteinase K with Catapult Buffer when necessary Laser microdissection and pressure catapulting Take an autoclaved cap tube Pipet 3 5 ul of Catapult Buffer into the middle of the cap optional method see below Put the cap tube into the cap tube holder Perform laser microdissection and laser pressure catapulting of selected cells or cell areas into the cap AUNE NOTE for frozen sections you can go on directly with 5 For formalin fixed material a Proteinase K digestion is necessary at that point see below 5 Remove the cap tube from the cap tube holder and put it onto a 0 5 ml microfuge tube
7. P A L M Microlaser Technologies PALM MicroLaser Systems Red blood cells Cut areas from a histological slide after catapulting found in the collection device Chromosome metaphase Selection of one chromosome PROTOCOLS for specimen preparations specimen isolation and collection downstream applications PALM MicroLaser Systems x PA L M Protocols Microlaser Technologies Protocols Specimen preparation Specimen isolation and collection Downstream applications Specimen preparatiOn cccccccceeeee eee eeeeeeeeee eee e ees 4 gt Preparation Or Slides sicctcccatesdesancsiaescnsasecsntsenetsacsaciaenhaenescnecel 4 e Samples on glass slides e Samples on membrane slides o UV treatment o Poly L Lysine treatment o Treatment to remove RNases gt Mounting sections onto slides isvesiiwssieivin Wiaierwiminisimiviwceiasrsen 5 e Paraffin embedded sections e Frozen sections e Cytospins e Blood and tissue smear gt Staining of the sections inca cence ositoscctatnereccneunasadeenceadeeuessadeaenes 6 e DEPC treatment of water and solutions e Histological staining methods o Hematoxylin Eosin H amp E HE Methylene Blue Methyl Green Nuclear Fast Red O O O Protocols_EN_0403 doc 2 14 o v PALM MicroLaser Systems amp PA L M Protocols Microlaser Technologies Specimen isolation and collection ccceeeeee eee 8 gt Laser cutting microdissection of the sampl
8. bation steps of histochemistry as short as possible Please use RNase free water and solutions e DEPC treatment of water and solutions RNases in aqueous liquids can be destroyed with 0 1 DEPC diethylpyrocarbonate CAUTION DEPC is highly toxic and should be handled under a fume hood Wear gloves 1 L of bidestilled H20 or solution 1 ml DEPC e g Roth K028 1 are stirred for 6 8 hours at room temperature then let incubate overnight in a fume hood The next day residual DEPC is removed by autoclaving Store treated solutions at room temperature NOTE All chemical substances containing amino groups e g TRIS MOPS EDTA HEPES etc cannot be treated directly with DEPC Prepare these solutions in DEPC treated H20 Protocols_EN_0403 doc 6 14 PALM MicroLaser Systems d PA L M Protocols Microlaser Technologies e Histological staining methods o Hematoxylin Eosin H amp E HE HE staining is used routinely in most histological laboratories and does not interfere with DNA and RNA preparation The nuclei are stained blue the cytoplasm pink red Procedure directly from distilled water 10 minutes Mayer s hematoxylin solution e g SIGMA MHS 32 10 minutes rinsing in running tap water 5 minutes Eosin Y e g SIGMA HT110 2 32 increasing ethanol series let air dry HE staining of Cryosections for RNA preparation directly from distilled water 3 minutes Mayer s Hematoxylin solution e g SIGMA MHS 32
9. containing lysis buffer or respectively pipet lysis buffer into the tube with the attached cap 6 Mix and lyse the cells by inversion 7 Centrifuge the sample at full speed for 5 minutes If using cryosections you can now go on straight forward with RNA extraction by using kits like the PALM RNA ExtractionKit 4600 0100 50 rct RNeasyMini Qiagen 74104 Absolutely RNA Nanoprep kit Stratagene 400753 High Pure RNA Tissue kit Roche 2033674 Purescript RNA isolation kit Gentra Distrib Biozym 212010 or your extraction procedure of choice The PALM RNA ExtractionKit is optimized for work with Laser Microdissection and Presure Catapulting LMPC If not going on directly store the samples in lysis buffer at 80 C If using paraffin sections it is necessary to perform a Proteinase K digestion step before starting the RNA extraction see below Protocols_EN_0403 doc 12 14 PALM MicroLaser Systems d PA L M Protocols Microlaser Technologies o Proteinase K digestion a After centrifugation 4 from above pipet 20 ul of Catapult Buffer containing Proteinase K onto the catapulted cells which are now in the tip of the tube b Vortex gently Digest for 2 18 hours at 55 C At best use a thermal cycler with a heating lid for the standard digestion c Centrifuge the sample at full speed for 5 minutes and then pipet lysis buffer into the tube Mix and lyse the cells by inversion d Incubate at 42 C for 30 min
10. energy is required for cutting Thus it is possible to perform laser ablation of unwanted specimen with moderate laser energy without immediate cutting the membrane After cleaning of the surrounding a higher laser energy is required to circumcise and catapult the selected specimen These slides are only available on request o UV treatment To overcome the hydrophobic nature of the membrane it is advisable to irradiate with UV light at 254 nm for 30 minutes The membrane gets more hydrophilic therefore the sections paraffine and cyrosections will obtain a better adherence Positive side effects are sterilization and destruction of potentially contaminating nucleic acids Protocols_EN_0403 doc 4 14 PALM MicroLaser Systems d PA L M Protocols Microlaser Technologies o Poly L Lysine treatment Additional coating with poly L Lysine 0 1 w v will be only necessary for special materials e g brain sections and should be performed by distributing a drop of the solution on the membrane Let it air dry at room temperature for 30 minutes Avoid any leakage underneath the membrane as this might result in impairment of Laser Pressure Catapulting o Treatment to remove RNases To ensure RNase free membrane slides we dip them for a few seconds into RNase ZAP Ambion 9780 250 ml followed by two separate washings in DEPC treated water see page 6 and drying at 37 C for 30 minutes up to 2 hours Subsequently the standard UV treatment
11. es cccceeeeee sean ees 8 e Tricks for improvement of the morphology sight of sections o Ethanol o Glycerol in PBS gt Laser Pressure Catapulting of the samples ccceeeeseeeeeeeeeees 9 gt Looking into the cap to see the catapulted samples 008 9 gt Getting the collected cells from the cap into the tip of the tube 9 Downstream Applications cccccceceee essence ee eeaeees 10 DNA ccliiccangcataraveienissridaiauand coniatvedartedeaiatananedenkesnideiensdatieaseeiacssne 10 gt Preparation of DNA from catapulted Samples cccccceeee sense eens ees 10 RNA eee cicsnces ouen vade omen excise sce aaree aa OA ORTON 11 gt Some special tips for working with RNA cccccecseceeseeeessereeseees 11 gt Preparation of frozen sections saci cccdccccasacnnacsascdnndanescucncersaaessess i gt Preparation of RNA from catapulted Samples cccceeeee sence tenn ees 12 o Proteinase K digestion Chromosome preparation on request cccccceee cece eens eens 14 Isolation of living cells on request cccccce cece e eee eee eeeeenaees 14 Protocols_EN_0403 doc 3 14 7 gt lt PALM MicroLaser Systems 4 PA L M Protocols Microlaser Technologies gt Preparation of slides When working with low magnifying objectives including 40x and 63x long distance objectives regular 1 mm thick glass slides can be used Due to the short working distance of the
12. ker Observe the area at the screen The depiction of the cells is improved immediately after having contact with the ethanol Now mark the cells or cell area with the software tool Ethanol is evaporating rapidly now catapult the marked cells or cell areas o Glycerol in PBS The same effect is produced by 1 glycerol in 1x PBS phospate buffered saline The time period until drying of the section is longer than with ethanol Handling of pipetting drying marking and cutting is the same as shown with ethanol above Protocols_EN_0403 doc 8 14 PALM MicroLaser Systems e PA L M Protocols Microlaser Technologies gt Laser Pressure Catapulting of the samples Catapulting into the cap Please have a look also in the PALM MicroBeam User Manual Pipette 3 to 5 ul bidistilled water buffer or light weight mineral oil PCR oil into the inner ring of the cap Be aware that aquaeus solutions will dry out after a while The catapulted cells or cell areas will stick onto the wet inner surface of the cap and will not fall down after the catapulting procedure For single cells or very small areas spinning down from oil may be difficult and therefore aquaeus solutions should be preferred When using membrane mounted samples the dissected membrane acts as a backbone for the selected area cell and can therefore be catapulted with a single laser shot from a remaining bridge at the border The morphological integrity is completely p
13. reserved with this procedure When using glass mounted samples it may be advisory to put more liquid up to 20 ul into the cap since the smaller flakes produced by multiple LPC points cannot be catapulted so straight to the centre of the cap as areas on membrane gt Looking into the cap to see the catapulted samples To control the efficiency of catapulting it is possible to look into the collection device e g PCR cap with the 5x 10x 40x and 63x objectives By using the software function go to checkpoint the slide is moved out of the light path and the cap can be lowered further towards the objectives for looking inside Normally most catapulted areas cells can be found within the small inner ring of the caps gt Getting the collected cells from the cap into the tip of the tube After microdissection the fluid is shortly spun down in a bench centrifuge 5 10 minutes 13000 rpm and samples can then be stored for later use For RNA extraction first appropriate lysis buffer is added to the PCR tube and after closure mixed by inversion Then the lysate is spun down as above for future RNA isolation analysis the tube is placed on ice or stored at 80 C in a freezer Protocols_EN_0403 doc 9 14 PALM MicroLaser Systems d PA L M Protocols Microlaser Technologies gt Preparation of DNA from catapulted samples For DNA prep from paraffin sections we usually use a Proteinase K con
14. see above is performed as usual NOTE Find special tips for work with RNA at page 11 Applications RNA gt Mounting sections onto slides Sections are mounted onto MembraneSlides the same way as routinely done with glass slides For cutting and catapulting a coverslip or standard mounting medium must not be applied e Paraffin embedded sections Mount the sections onto the slide as routinely done Afterwards let dry at 37 C up to 56 C overnight in a drying oven Deparaffinization Paraffin will reduce the efficiency of laser cutting sometimes it will make it impossible to cut and catapult If working with unstained sections it is therefore very important not to forget removing the paraffin before laser cutting and laser pressure catapulting If applying staining procedures deparaffinization is routinely included in any protocols Procedure Xylene 2 times for 2 minutes Ethanol 100 1 minute Ethanol 96 1 minute Ethanol 70 1 minute rinse with water Prolonged Xylene treatment could resolve membrane fixation of POL slides Xylene treatment should therefore be kept as short as possible With the PALM MembraneSlides 1440 1000 it is possible to extend the time of Xylene treatment up to 15 minutes Protocols_EN_0403 doc 5 14 PALM MicroLaser Systems z PA L M Protocols Microlaser Technologies e Frozen sections Fixation After mounting the sections there are many possibilities to fix the material If
15. taining catapult buffer This step is not necessary for frozen sections Catapult Buffer 0 5 M EDTA pH 8 0 20 ul 1 M Tris pH 8 0 200 ul Igepal CA 630 SIGMA I 3021 50 ul Proteinase K 20 mg ml 100 ul ddH20 fill up to 10 ml Proteinase K Lyophilizate Roche Mannheim Germany Catalog 1413783 or Proteinase K Solution 20 mg ml Qiagen GmbH Hilden Germany Catalog 19131 Always prepare a fresh mixture of Catapult Buffer and Proteinase K Take an autoclaved cap or tube Pipet 3 5 ul of Catapult Buffer PCR oil or DNase free water in the middle of the cap Put the cap tube into the cap tube holder Perform laser microdissection and laser pressure catapulting of selected cells or cell areas Remove the cap from the cap holder and put it onto a new 0 5 ml microfuge tube or just close the used tube with the attached cap Centrifuge the tube at full speed for 5 10 minutes 7 Add 10 15 ul Catapult Buffer containing Proteinase K onto the cells which are now in the tip of the tube 8 Proteinase K digestion a Vortex gently Digest for 2 18 hours at 55 C followed by a heating step at 90 C for 10 min to inactivate Proteinase K Optional and recommendable for AutoLPC Catapult directly into 20 ul Catapult Buffer containing Proteinase K Digest upside down in the cap for 2 18 hours at 55 C in an incubator After centrifugation inactivate Proteinase K for 10 minutes at 90 C b At best use a thermal cycler
16. tion since many factors like species cell tissue type degradation fixation staining fragmentation extraction procedure and others will influence the outcome From mouse liver frozen sections we can roughly retrieve 5 20 pg of RNA per cell calculated from extractions of 1000 cells and analysis on an Agilent Bioanalyzer see also Agilent Application Note 5988 EN on our homepage or at www chem agilent com The time necessary for complete digestion depends on the kind and on the number of catapulted cells Protocols_EN_0403 doc 13 14 PALM MicroLaser Systems e PA L M Protocols Microlaser Technologies Chromosome preparation Protocols on request Isolation of living cells Protocols on request Please note There is also a review brochure available dealing with live cell laser micromanipulation PALM Scientific Edition No 11 ISBN No 3 9808893 0 0 For questions remarks or protocol requests please contact P A L M s Service amp ApplicationLaboratory Email Servicelab palm microlaser com Service Line 49 0 81 58 99 71 300 Protocols_EN_0403 doc 14 14
17. to improve the lysis e Centrifuge the sample at full speed for 5 minutes Now proceed with the RNA extraction see above Optional method also recommendable for AutoLPC a Instead of step 2 from above catapult directly into 20 ul of Catapult Buffer containing Proteinase K b Digest upside down in the cap for 2 18 hours at 55 C in an incubator c Centrifuge the sample at full speed for 5 minutes and then pipet lysis buffer into the tube Mix and lyse the cells by inversion d Incubate at 42 C for 30 min to improve the lysis e Centrifuge the sample at full speed for 5 minutes Now proceed with the RNA extraction using kits or your extraction procedure see above If not going on immediately store the samples at 80 C in lysis buffer P A L M Procedure For best RNA quality we use frozen sections on PALM MembraneSlides and perform catapulting of the cells directly into RNase free water containing 0 5 Igepal or into Catapult Buffer without Proteinase K After catapulting the samples are mixed with lysis buffer as soon as possible to protect the RNA NOTE If using paraffin sections for catapulting please use a buffer containing Proteinase K Best use freshly cut specimens Frozen sections should not be stored for more than a few days at 80 C Freezing should be performed after ethanol fixation or after staining and drying It is difficult to estimate the amount of RNA that is to be expected after extrac
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