Patsy, If the animal was treated with BrdU initially, then yes I have. Unfortunately All of my papers and reference materials are boxed up at the moment (preparing to move) but I have done this procedure so many times that I can pretty much reconstruct it from memory.
1) Hydrate slides to ddH2O as per SOP. 2) Hydrolyze with 4N HCl at 37oC for 20 minutes. 3) Rinse with ddH2O for 1 minute at RT. 4) Transfer slides to a staining dish filled with ddH2O (kept at 37oC) for 5 minutes. 5) Incubate in pepsin solution (Dako) for 15 minutes at 37oC. All of the remaining steps are at room temp. 6) Rinse 2X with ddH2O, 1 minute each rinse. 7) Rinse 2X with PBSt, 3 minutes each rinse 8) Peroxidase Blocking Reagent (Dako), 5 minute incubation. 9) Repeat step #7. 10) Primary antibody (anti-BrdU), incubate 10 minutes at a 1:200 dilution. [5 μL antibody to 995 μL 1% BSA in PBS] 11) Repeat step #7. 12) Polymer labeled secondary antibody (Dako Envision HRP), incubate for 10 minutes. 13) Repeat step #7. 14) Incubate in working Dako DAB solution for 8 minutes (1 drop DAB Chromogen per 1 ml substrate buffer). 15) Rinse well with ddH2O. 16) DAB enhancer solution (2.5% CoCl2 , 2.0% NiSO4(NH4)2SO4 in ddH2O) for 8 minutes 17) Repeat step #15. 18) Richard Allan Hematoxylin 1 for 25 seconds, rinse with tap water until there is no blue color in the water. 19) Let sit in tap water for 5 minutes. 20) dehydrate and coverslip in accordance with SOP’s. Best of luck, Bob Schoonhoven ________________________________ From: Patsy Ruegg <pru...@ihctech.net> To: histonet@lists.utsouthwestern.edu Sent: Friday, April 17, 2009 1:26:36 PM Subject: [Histonet] brdu on ffpe rat fixed for up to 2 years in NBF Anybody tried doing IHC for brdu on ffpe rat intestine and skin fixed for up to 2 years in NBF? Thank you, Patsy Patsy Ruegg, HT(ASCP)QIHC IHCtech 12635 Montview Blvd. Ste.215 Aurora, CO 80045 720-859-4060 fax 720-859-4110 www.ihctech.net www.ihcrg.org _______________________________________________ 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