Re: [Histonet] RE: Frozen section fixing problems - but paraffin works!
Chris van der Loos's suggestions are superb. You can buy a peroxidase
block from Dako that is designed for frozen sections, Perox Block, S2001 -
with sodium azide, in a buffer with 0.03% hydrogen peroxide. I works very
well without chewing your sections off the slide, ready to use in a dropper
bottle. and we use it for convenience with most of our frozen section
animal peroxidase IHC.
Drying frozen sections is critical! After acetone fixation, we go in front
of the fan for 20 mins or so, to insure no residual acetone is present,
sections are dry.
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
At 12:34 PM 11/20/2003 +0100, you wrote:
>Your protocol for staining the cryostat sections needs some adaptions
>First, cut your sections and allow them to dry at least 1 hour at RT.
>Preferably longer, for example over night under a fan. Next, fix your
>cryo's in pure acetone for 10 min at 4C, let them air-dry. Block
>endogenous peroxidase activity with 0.3% perhydrol + 0.1% Na-azide in
>TBS or PBS (20 min, RT) and wash with TBS or PBS (3 x 2 min). Then,
>start with the donkey serum etc. and your usual staining procedure.
>You should avoid the use of alcohols or methanol in the staining
>procedure, because most antigens will be damaged by this.
>Blocking with 0.3% peroxide (in buffer I hope??) is not recommended
>because the formation of air bubbles that may damage your tissue
>section. The addition of Na-azide prevents the formation of air bubbles
>and also acts as inhibitor of peroxidase activity.
>Good luck with staining!
>Chris van der Loos
>Dept. of Cardiovascular Pathology
>Academical Medical Center
>Amsterdam - The Netherlands
>----- Original Message -----
>>From "Tan, MinHan"
>Date Wed, 19 Nov 2003 17:32:59 -0500
>Subject [Histonet] Frozen section fixing problems - but paraffin
>I am working with a new monoclonal antibody in parathyroid tissue, and
>I use the ABC-DAB reageant system for immunohistochemistry.
>I am new to frozen sections immunostaining - and I am encountering
>problems with frozen sections - staining is very weak or absent, in
>comparison to paraffin embedded tissue, which I have no problems with.
>My protocol for frozen sections:
>Unfixed frozen slides with 5 micron sections thawed at room temperature
>for 5 minutes
>Placed in 70% ethanol x 5 minutes.
>Washed in PBS x 5 minutes.
>followed with standard protocol: hydrogen peroxide 0.3% x 30 min,
>donkey serum 5% x 30 min, primary antibody (mouse) 4 deg overnight,
>secondary (goat anti-mouse), ABC, DAB.
>Does anyone have any advice?
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