[Histonet] Cytoplasmic staining with Ki67 in frozen sections

[Liz Chlipala]

James

I believe the nuclear staining pattern that you describe is normal for
Ki-67 in the spleen, not sure about the cytoplasmic or particulate
staining.  

Since you viewing with fluorescence the particulate staining may be
autofluorescence of red blood cells. Normally our primary antibody
incubations are only 30 minutes, 2 hours at RT seems a bit much. Did you
try multiple dilutions of the primary?

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of James S.
Sent: Thursday, January 27, 2011 2:36 AM
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] Cytoplasmic staining with Ki67 in frozen sections

HI All,

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!!

Any suggestions?

Thanks
Sonya


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