IHC help please - brown everywhere
|From:||"J.L. Turnbull" <firstname.lastname@example.org>|
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
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
Please help (and sorry it's not more concise)
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