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<DIV>Kathleen,</DIV>
<DIV> </DIV>
<DIV>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.</DIV>
<DIV> </DIV>
<DIV>Clean glass slides with 95% ethanol, treat with subbing solution and air
dry (not necessary to sub, if using SuperFrost Plus slides).</DIV>
<DIV>Cut 4-8 micron thick sections of tissue block.</DIV>
<DIV>Allow frozen sections to come to room temp. </DIV>
<DIV>Fix slides with cold acetone for 10 minutes (I've used 4% Formaldehyde and
have gotten great results).</DIV>
<DIV>Rinse in three changes of Buffer.</DIV>
<DIV>Incubate for 5 to 10 minutes in 0.1% hydrogen peroxide in PBS to quench
endogenous peroxidase activity.</DIV>
<DIV>Rinse with PBS</DIV>
<DIV>Incubate slides with blocking serum in PBS for 20 minutes, to suppress
non-specific binding of immunoglobulin.</DIV>
<DIV>Rinse with three changes of PBS, 5 minutes each rinse.</DIV>
<DIV>Incubate with Primary antibody for one hour at room temp or overnight at 4
degrees C.</DIV>
<DIV>Rinse three times with PBS as above</DIV>
<DIV>Incubate with secondary antibody (usually biotinylated) for 30
minutes</DIV>
<DIV>Rinse three times</DIV>
<DIV>(In this protocol they use ABC reagent)</DIV>
<DIV>Incubate with ABC Reagent for 30 minutes</DIV>
<DIV>Wash three times with PBS</DIV>
<DIV>Rinse with 1% Triton X-100/PBS for 30 seconds</DIV>
<DIV>Incubate with DAB solution for 1-3 minutes</DIV>
<DIV>Rinse with distilled water</DIV>
<DIV>Counterstain (if desired) with Hematoxylin for 1-3 minutes</DIV>
<DIV>Rinse with distilled water</DIV>
<DIV>Dehydrate, Clear with Xylene and mount with ageous mounting
medium.</DIV>
<DIV> </DIV>
<DIV>I hope this helps, if it's totally off the mark for you, sorry.
</DIV>
<DIV>Gareth</DIV>
<DIV> </DIV>
<DIV> </DIV>
<DIV> </DIV>
<DIV><FONT face=System color=#800080 size=2>Ms. Gareth B. Davis</FONT></DIV>
<DIV><FONT face=System color=#800080 size=2><STRONG>Research Assistant
II</STRONG></FONT></DIV>
<DIV><FONT face=System color=#800080 size=2>Neuro-magnetics Division of
</FONT></DIV>
<DIV><FONT face=System color=#800080 size=2>the Department of
Neurology</FONT></DIV>
<DIV><FONT face=System color=#800080 size=2>Vanderbilt University Medical
Center</FONT></DIV>
<DIV><FONT face=System color=#800080 size=2>615-936-3318</FONT></DIV>
<DIV><FONT face=System color=#800080 size=2></FONT> </DIV>
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