Your question leads me to believe you are using an antibody that you
have used previously with good results and a direct HRP tagged
secondary. If this is indeed the case you should be able to adjust the
concentrations so that both secondaries have the same concentration. It
would be necessary to include a no primary control to determine the
amount of fluorescence due to the tagged secondary. I like to do a
control for auto fluorescence as well.

I did not hear any reference to a known positive or negative control
those would have an impact on determining the protocol as well. 

I personally would cut a bit thinner 6-8um.

c

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317

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-----Original Message-----
From: Donin, Nick (NIH/NCI) 
Sent: Monday, October 24, 2005 8:51 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Frozen Section Protocol

Histonet, 

My PI has requested that I section and stain sections of a subcutaneous
human tumor that is frozen.  He wants me to cut 10 micron sections, and
then stain the tumor and look at it using fluorescence (as opposed to
some chromogen technique like Vectastain).  Can anyone suggest a
protocol?  Below is the protocol I have been using so far, with poor
results (high background, very little specific staining.  Thanks
everyone. 

 

Freeze tumor in isopentane - 5 seconds

Section tumor at 10 microns and place on slide

Wash 1x with PBS

Fix 15 min with 4% PFA 

Block 1 hr with 5% NGS with 0.3% Triton

Incubate 1 hr RT with primary antibody

Wash 3x 5 min with PBS

Incubate 1 hr RT with secondary antibody

Wash 3x 5 min with PBS

Apply Dapi and coverslip 

(all staining and washing is done by covering tissue with solution while
tissue is on the glass slide)

 

Nick Donin

CRTA

Neuro-Oncology Branch

National Cancer Institute

National Institutes of Health

9000 Rockville Pike

Building 35, Room 2B-203

Bethesda, MD 20892

 

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