IHC help please - brown everywhere -Reply

From:Tony Henwood <AnthonyH@chw.edu.au>

You could try checking your concentration of hydrogen Peroxide in you
DAB reagent. Too concentrated and you will end up with rapid
nonspecific DAB deposition.
Tony Henwood JP, BappSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at  Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: (02) 9845 3306
Fax: (02) 9845 3318

>>> "J.L. Turnbull" <jlt25@hermes.cam.ac.uk> 20/April/2001 01:06am >>>

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
sections in paraffin at 56 degrees.  I rehydrated by standard
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
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,

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
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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