Guillermo We have not used that kit specifically we use the one from Roche but we get fine results with the following fixatives, 10% NBF, 4% Paraformaldehyde and Davidisons, its also works fixed and EDTA decalcified samples. You will need to add a proteinase K step to your protocol. You need to order a specific proteinase K the one you may use for IHC may not work. We get ours from Roche here is the catalog number and how we prepare the stock and working solutions, you may need to titer the concentration of proteinase K for your particular project we use it at a concentration of 20ug/ml. I have also added our basic protocol so you can see where the protienase K step figures in.
Proteinase K, recombinant, PCR Grade Roche Applied Science 03 115 879 001 Proteinase K Stock Solution - 20mg/ml Proteinase K 1 vial - 100mg Distilled water 5 mls Once the proteinase K lyophilized enzyme has been reconstituted with distilled water then aliquot into RNase free 0.6 ml microtubes a volume of 20 to 40µls and store at -15 to -25°C. Shelf Life: 12 months Storage: -15 to -25°C Proteinase K Working Solution - 20ug/ml For each slide that needs to be stained you will need to prepare a minimum of 125µls of proteinase K working solution. 100µls of working solution will be placed on each slide. The working solution is prepared as follows: Proteinase K Stock Solution 10µls 10Mm TRIS/HCL pH8.0 990µls Make fresh prior to use. Remove one aliquot vial of the stock 20mg/ml proteinase K from the freezer and thaw. Unused stock proteinase K may be stored in the refrigerator for 7 days, then discard. Shelf Life: make fresh prior to use, discard unused portion Storage: NA Procedure When performing the TUNEL stain the maximum number of slides that can be run at a time is 12. Within the 12 slides, 10 will be test samples, 1 is a positive control (DNase treated) and 1 is a negative control (Label Solution without Enzyme Solution). All but the rinse steps are performed either in a humidity chamber or in the Dako Hybridizer. 1. Xylene 5 minutes 2. Xylene 5 minutes 3. 100% alcohol 2 minutes 4. 100% alcohol 2 minutes 5. 95% alcohol 1 minute 6. 95% alcohol 1 minute 7. Tap water rinse 1 minute 8. Distilled water rinse 1 minute 9. PBS pH7.4 0.1% tween rinse 1 minute or more 10. Prepare working proteinase K - see solutions 11. Working proteinase K (75 -200µls/slide) 30 minutes 12. PBS pH7.4 0.1% tween rinse 3 minutes 13. PBS pH7.4 0.1% tween rinse 3 minutes 14. Prepare DNase1 working solution - see solutions 15. DNase1 (75 -150µls/slide) 10 minutes Note: apply DNase1 to only the positive control sample, the rest of the samples will remain in PBS pH7.4 0.1% tween buffer 16. PBS pH7.4 0.1% tween rinse 3 minutes 17. PBS pH7.4 0.1% tween rinse 3 minutes 18. Prepare TUNEL reaction mixture Note: The TUNEL reaction mixture should be prepared immediately before use. Keep TUNEL reaction mixture on ice until use. Unused reagent may be stored at -70°C for future use. Prepare the TUNEL reaction mixture and negative control solution as follows: a. The TUNEL kit reagents are stored in the -70°C freezer b. Remove one vial 1: enzyme solution and one vial 2: label solution from the freezer c. Remove 100µl of the label solution and place it into a microtube this solution will serve as the negative control solution d. Add the total volume of vial 1: enzyme solution (50µl) to the remaining 450µl in vial 2: label solution and mix well 19. Remove slides from PBS and dry area around sample 20. Add 50 to 100µl of TUNEL reaction mixture 21. Incubate at 37°C in the Dako Hybridizer 60 minutes NOTE: for the negative control add 50 to 75µl of the label solution to each sample. If necessary to ensure a homogenous spread of TUNEL reaction mixture across the sample and to avoid evaporative loss, the samples can be covered with a coverslip during incubation. 22. PBS pH7.4 0.1% tween rinse 3 minutes 23. PBS pH7.4 0.1% tween rinse 3 minutes 24. PBS pH7.4 0.1% tween rinse 1 minute 25. Add 50 to 100µl of Converter-AP (vial 3) to each sample 26. Incubate at 37°C in the Dako Hybridizer 30 minutes NOTE: If necessary to ensure a homogenous spread of TUNEL reaction mixture across the sample and to avoid evaporative loss, the samples can be covered with a coverslip during incubation. 27. PBS pH7.4 0.1% tween rinse 3 minutes or more 28. PBS pH7.4 0.1% tween rinse 3 minutes or more 29. Wash Buffer 1 minute 30. Prepare Vulcan Fast Red Substrate Solution To 2.5 mls of Vulcan Fast Red Buffer add one drop of vial A and one drop of vial b. Use immediately, discard any leftover reagent. 31. Vulcan Fast Red Substrate Solution 10 - 30 minutes 32. Distilled water 2 minutes 33. Distilled water 2 minutes 34. Hematoxylin 3 minutes 35. Distilled water 1 minute 36. Wash Buffer 1 minute 37. Distilled water 1 minute 38. Let slides air dry 39. Xylene 1 minute 40. Mount and coverslip 41. Review positive and negative control slides for appropriate staining Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Manager Premier Laboratory, LLC P.O. Box 18592 Boulder, CO 80308 phone (303) 682-3949 fax (303) 682-9060 l...@premierlab.com www.premierlab.com Ship to Address: Premier Laboratory, LLC 1567 Skyway Drive Unit E Longmont, CO 80504 -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Guillermo Palchik Sent: Tuesday, December 16, 2008 3:37 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] TUNEL on FIXED rat PUPS Dear Histologists... I am currently doing an experiment in which I perform TUNEL on brain slices that were treated with certain drugs, to examine the levels of apoptotic cell death. The rat's brain is flash frozen (scooped from the skull, directly into cold isopentane) air dried, and then stored into a -80 C freezer. We then cut the brain into 20 um slices using a cryostat and mount them on slides, and store them in a -20 C freezer, where they remain until the TUNEL step. The TUNEL is performed using the Apoptag Plus kit from Chemicon using Peroxidase/DAB (Cat. # S7101) and counterstaining with 0.5% methyl green. This has been done in the lab for quite some time now and we are able to get results... We want to switch over to perfused brains (instead of flash frozen). However, we have had ZERO positive staining once the brains are fixed (in 4% PF). This has been corroborated by other lab members that have tried it for a couple of years already... The protocol that came with the kit has a section for flash frozen and a section for fixed tissue, and since I have done TUNEL using fixed tissue before, I know that it is possible to do TUNEL in fixed tissue, however we cannot get any positive staining whatsoever... Along these lines, since the first step of the Apoptag TUNEL protocol is to fix the tissue with 4% PF (for 10 min), this has led me to believe that the problem is indeed the initial fixation with PF (at the time of perfusion). I should say that we work with rat PUPS for this and that the immature brain is not the same as the mature brain (the immature brain has more fat, for example) and that this might be the cause of the problem...In any case, I was doing some research and I wanted to try using Zinc Formalin as a perfusate and see if this would allow us to do the TUNEL. I would appreciate any comments and suggestions regarding this. I am sorry for the lengthy email, but I wanted to show a more or less complete picture, in case I am overlooking other factors... Guillermo Palchik g...@georgetown.edu _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet