[Cytometry] Ethanol fixation and surface staining

SIMON MONARD smonard at staffmail.ed.ac.uk
Fri Mar 27 11:29:53 EDT 2009


Hi Simona

I've used the BD lysing solution after surface staining to  
permeabilize cells prior to Ki67 staining with some success. Give it  
15-30 mins or so and wash a couple of times before the Ki67 ab. I  
always found ethanol spoils surface staining.

Simon

Quoting "McClellan, Steve" <smcclell at ufl.edu>:

> Hi Simona,
> I recently validated a protocol using Ki-67 combined with surface   
> markers to monitor engraftment/subset proliferation after BM   
> transplant in a mouse model.  BD has tested many cell surface   
> antibodies for compatibility with ethanol for their phospho-specific  
>  antibodies.  You can find the list at   
> http://www.bdbiosciences.com/features/products/display_product.php?keyID=94#add . I only used antibodies that were on the list. If you use others it will be trial and error on your part to find ones that work.  Many epitopes are not stable with ethanol, sometimes there are even clone differences between antibodies for a given marker.  I did not have much success using paraformaldehyde/saponin to get reproducible Ki-67 staining, best to stick to   
> ethanol.
> Good luck,
> Steve
>
> Steve McClellan
> Sr. Biological Scientist
> Interdiciplinary Center for Biotechnology Research(ICBR)-
> Flow Cytometry Core Laboratory
> University of Florida
> 352-273-8185 (office)
> 352-273-8070 (fax)
> smcclell at biotech.ufl.edu
>
>
>
>
>
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu   
> [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Simona Zompi
> Sent: Tuesday, March 24, 2009 1:41 PM
> To: cytometry at lists.purdue.edu
> Subject: [Cytometry] Ethanol fixation and surface staining
>
> HI everyone
> I have done an intracellular staining for Ki67 using the BD protocol after
> staining extracellularly for several markers (CD138, IgD, B220, CD79b)
>
> Fixation/perm protocol is as follow:
> 70% Ethanol fixation for at least 2h at -20°C, 2 washes then staining with
> Ki67 20 min RT in PBS+BSA then 2 washes.
>
> I have run the cells the day after as it was quite late and stored them at
> 4°C overnight.
>
> Intracellular was great but I had lost some of the extracellular markers
> (mainly CD138).
>
> Do you have any suggestions? Did it happen to you? Could I use the usual
> PFA+Saponin fix/perm for anti-Ki67?
>
> Thank you for your help.
>
> Simona ZOMPI
> School of Public Health
> UC Berkeley
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Simon Monard
FACS Facility Manager
MRC Centre for Regenerative Medicine
Institute for Stem Cell Research
University of Edinburgh
Roger Land Building
West Mains Road
Edinburgh
EH9 3JQ

Tel. Lab 0131 6505876
Tel Office 0131 6517265

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