0764 v1.0 Duolink II with Shandon Guidelines - final2
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1. 752 37 Uppsala Sweden www olink com2. Duolink II GUIDELINES 0764 v1 0 Using Duolink Il with Shandon Sequenza immunostaining system RECOMMENDED MODIFICATIONS TO THE DUOLINK II FLUORESCENCE USER MANUAL DOC NO 0650 OR DUOLINK II BRIGHTFIELD USER MANUAL DOC NO 0656 When using the Duolink Il reagents to stain samples with the Shandon Sequenza immunostaining system a few modifications to the Duolink II protocol optimized for manual staining of samples deposited on glass slides have to be made in order to suit this system In particular the primary antibody concentrations and reagent and washing volume differ from the standard protocol We recommend you to carefully read through the User Manual Doc no 0650 or 0656 and adjust the protocol according to the following guidelines when combining Duolink II with the Shandon Sequenza immunostaining system Additional modifications to the protocol might also be necessary to meet your specific application or assay design RECOMMENDED EQUIPMENT gt Shandon Sequenza slide rack b Shandon Sequenza disposable coverplates b Microscope slides with fixed cells or tissue Note Do not use slides where the sample is delimited by a well or hydrophobic barrier Fisher Scientific art no 73310017 t Fisher Scientific art no 72110017 case of 25 art no 7219950 case of 50 art no 72110013 case of 250 PRE TREATMENT BLOCKING AND PRIMARY ANTIBODIES The conditions for your primary antibodies should be optimized with r
3. espect to sample fixation antigen retrieval blocking solution antibody diluent and incubation temperature and time Note You will need a This product is for research use only 2011 Olink AB Duolink and PLA are trademarks of Olink AB All third party trademarks are the property of their respective owners OLINK BIOSCIENCE higher concentration of the primary antibodies with the Shandon Sequenza system compared to when using Duolink II for manual staining to receive a comparable number of signals All steps starting from blocking to the Final Wash Step should be performed when the slides are mounted to the Shandon Sequenza coverplates and placed in the slide rack When mounting the slide to the coverplate add 500 ul of PBS or sample buffer to the coverplate to create a liquid surface between the slide and the coverplate INCUBATION STEPS 100 ul of reagents per slide is recommended All Duolink Il reagent stocks should be diluted to the concentrations described in the User Manual During the 37 C incubations put the lid on top to prevent drying of the samples WASH STEPS Follow the washing recommendations in the User Manual but add 1000 ul of wash buffer slide per wash step e g during a 2x5 min wash add 1000 ul of wash buffer slide wait for 5 min then add another 1000 ul of wash buffer slide and wait for 5 min before you add new reagents 0764 v1 0 2011 02 03 Olink Bioscience Dag Hammarskj lds v 52B SE
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