[Histonet] Re: Protocol for frozen IHC

[Davis, Gareth]

Kathleen,
 
Here is a protocol from Oncogene Research.  I'm not sure where you need the protocol to start, so this starts from the very beginning.
 
Clean glass slides with 95% ethanol, treat with subbing solution and air dry (not necessary to sub, if using SuperFrost Plus slides).
Cut 4-8 micron thick sections of tissue block.
Allow frozen sections to come to room temp. 
Fix slides with cold acetone for 10 minutes (I've used 4% Formaldehyde and have gotten great results).
Rinse in three changes of Buffer.
Incubate for 5 to 10 minutes in 0.1% hydrogen peroxide in PBS to quench endogenous peroxidase activity.
Rinse with PBS
Incubate slides with blocking serum in PBS for 20 minutes, to suppress non-specific binding of immunoglobulin.
Rinse with three changes of PBS, 5 minutes each rinse.
Incubate with Primary antibody for one hour at room temp or overnight at 4 degrees C.
Rinse three times with PBS as above
Incubate with secondary antibody (usually biotinylated) for 30 minutes
Rinse three times
(In this protocol they use ABC reagent)
Incubate with ABC Reagent for 30 minutes
Wash three times with PBS
Rinse with 1% Triton X-100/PBS  for 30 seconds
Incubate with DAB solution for 1-3 minutes
Rinse with distilled water
Counterstain (if desired) with Hematoxylin for 1-3 minutes
Rinse with distilled water
Dehydrate, Clear with Xylene  and mount with ageous mounting medium.
 
I hope this helps, if it's totally off the mark for you, sorry.  
Gareth
 
 
 
Ms. Gareth B. Davis
Research Assistant II
Neuro-magnetics Division of 
the Department of Neurology
Vanderbilt University Medical Center
615-936-3318
 
 
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