[Histonet] Frozen Section Protocol

[Donin, Nick (NIH/NCI)]

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