[Histonet] Frozen Section Protocol
Favara, Cynthia (NIH/NIAID)
cfavara <@t> niaid.nih.gov
Mon Oct 24 11:26:08 CDT 2005
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 <@t> 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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