Summary Document

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Thank you for the answers, we will try different protocols.

	From:	Julie Bertout <julie.bertout@ibl.fr>
	To:	Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
	Subject:	Nuclear Receptor Staining : summary of answers
	Date sent:	Tue, 13 Nov 2007 13:12:06 +0100

Original Question:

Does anybody have a good protocol for nuclear receptor staining? Is
0,01% saponin permeabilisation enough to reach them or do we need to
change our regular protocol?
What are the best controls (positive and negative) to validate the
staining?

Responses:

I have use saponin permabilisation for staining PCNA and Ki-67 which are
nuclear antigens for several years now. Works perfect with standard 30
minute incubation time. I use stimulated and unstimulated lymphocytes as
pos/neg controls. We perform the surface stainig first and then the
permabilisation.
Kasia
kruck@amg.gda.pl

I do intranuclear Ki67 staining just using PFA for fixation and 0.5%
Saponin in PBS/FCS for permeabilization. We do not need anything else to
get good staining. You can also use the Cytofix/Cytoperm and Permwash from BD
with no problems..
Anne
Anne.Wilson@isrec.ch

You didn't inform us which protein you want to detect but we prefer
buffered freshly prepared 1.0% paraformaldehyde, 5 min on ice follwoed
by 70 - 90% ice cold methanol. Excelent for Ki67, PCNA p53, cyclins.
Willem Corver
W.E.Corver@lumc.nl

We have been performing nuclear antigens staining (not receptors though)
with and easy protocol consisting on 4% PFA fixing and permeabilizing
with eBiosciences buffer afterwards. Generally, we incubate the mAbs
with the buffer for 30min at RT and it looks like it's working very
nicely. There are reports on variability regarding the perm buffers
(eBiosciences vs BD vs caltag) as weel as fix solutions (methanol vs PFA).
Espe
esperanza.perucha@gmail.com