[Histonet] RE: Cytoplasmic staining with Ki67 in frozen sections
C.M. van der Loos
c.m.vanderloos <@t> amc.uva.nl
Thu Jan 27 12:07:57 CST 2011
Dear Sonya,Acetone is an excellent fixative for many antibody to stain frozens, however nuclear antigens like Ki67 after acetone fixation get a bit fuzzy. I would strongly recommend to fix the slides 5 min at room temp in standard 4% buffered formalin, wash with PBS (or TBS) and start your IHC. You will see a much sharper staining result that is more confined to the nucleus.Hope this helps.Cheers, ChrisChris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
NL-1105 AZ Amsterdam
Date: Thu, 27 Jan 2011 09:36:29 +0000
From: "James S." <sonya.martin <@t> soton.ac.uk>
Subject: [Histonet] Cytoplasmic staining with Ki67 in frozen sections
To: "'histonet <@t> lists.utsouthwestern.edu'"
histonet <@t> lists.utsouthwestern.edu
I've just been trying out a Ki67 antibody from Abcam (rabbit polyclonal) on some frozen mouse tissues. Staining looked ok in the colon with nuclear localisation in the cells of the basement membrane. However, what I'm really interested in is the spleen and the staining here was a mess! There was some nuclear staining especially in germinal centres and scattered around the white pulp but the red pulp contained clusters of cells with cytoplasmic staining only (I don't know if this is real staining, non-specific staining or an artefact), also there was bright particulate 'staining' all over the red pulp but not the white.
Frozen sections were cut (10um), air dried overnight, fixed in acetone (10mins), blocked with 5% normal goat serum, Ki67 Ab for 2hrs room temp, secondary was goat anti rabbit AlexaFluor 488 (Invitrogen - clean on its own).
I thought this Ki67 staining was going to be easy!!
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