IHC help please - brown everywhere

From:"J.L. Turnbull" <jlt25@hermes.cam.ac.uk>

I am a biochemist by trade, but ahve wentured into some histology, rather
unsuccessfully.  None of my biochemist contacts know anything about about this
so i'd really appreaciate any assistance.

I have been trying to locate protein insecticides in sections of
caterpillar gut with a  peroxidase-conjugated antibody.  I've done quite
a few runs and changed many variables - still brown.

I based my protcol on a published paper using similar insects.
I fixed in 2.5% gluteraldehyde 0.5M cacodylate for 24 hours.  Anatomy made
sections in paraffin at 56 degrees.  I rehydrated by standard techniquwes
and continued roughly as follows:

Incubate in iodine/K iodide

Equilibrate in Tris, Saline, Triton buffer.

Antigen revival in 1 mg/ml trypsin 10 min.

Quench endogenous peroxidase with 0.03% H2O2 in methanol 20 min.

primary monoclonal antibody in either milk. BSA, Haemoglobin or pre-immune
serum.  Washed many times.

Either peroxidase-conjugated secondary antibody or VECTASTAIN ABC
avidin-biotin kit.

Developed in either DAB or chloronaphthol in methanol,

Everything was brown, even antigen-free and antiserum-free controls.

1) the protcol said that the iodine step was to remove the sublimate, what

2) How does the quenching step work?  I used the same recipies plus
chromogen to develop and it didn't quench that?  Is it just oxidative

3)  I guessed that endogenous peroxidase was a problem (so haem. was bad
choice then i realise).  I tried using sodium azide as an inhibitor (is it
reversible or not?)  this didn't work either.

4) i didn't know i had to filter the DAB - could this be the explanation?
However. chloronapthol didn't work either.

5) the other protocol used Hollandes fixative - would this make a

6) I tried doing this with whole guts (whilst i made more slides) just to
see if the sectioning process had done something.  Most went brown but the
2 that i hadn't fixed gave a white control and brown sample.  Did i use
the wrong fixative therefore.  I'm looking mainly at membranes, so i
suppose methanol would have been better (I'm only just learning this
histology business)

Please help (and sorry it's not more concise)


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