Hi everyone, Sorry to keep posting about this,
But I am still having Acetone issues. I am doing IHC for Cell surface markers that are lost when fixing with etoh or methanol. When I fix in 100% acetone my epitopes have great signal. However, when I fix in 100% acetone, my tissues all damaged by the acetone beyond all recognition. I can lose up to 90% of my tissue when I fix in 100% acetone for 10 minutes. But, I get good epitope staining if I have some tissue left on the slide. When I fix in anything else, I lose 90% or more of my epitope staining, but my tissue morphology looks great. -- What's the least amount of time I can fix in 100% Acetone for a 5uM section and still have it be fixed? Is drying after the 100% acetone fixing essential? or Bad for protecting tissue morphology? -- I am doing IHC on human tonsils cut to 5 uM with a 24 dry after sectioning. When I fix in 100% acetone, I fix it at 4C for 10 minutes. Then dry for 1 hour in the fume hood Should it go straight into buffer? Should it be for less time in the acetone? Should the acetone be Room temp or -20C instead of 4C. If I was diluting the acetone with buffer, (or etoh) then I could see going straight into buffer afterwards, but because I am using 100% I think that going into liquid right after fixing is too much of a change and my tissues go BOOM. Please help. Patrick Lewis Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
