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 www.premierlab.com Ship to Address: 1567 Skyway Drive, Unit E Longmont, Colorado 80504 -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of James S. Sent: Thursday, January 27, 2011 2:36 AM To: 'histonet@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 _______________________________________________ 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