Re: IHC help please - brown everywhere

From:Greg Dobbin <dobbin@Upei.CA>

Hi Jenny,

First, the quench in H2O2 is to quench or "use up" endogenous 
peroxidases so that the substrate is only catalyzed by the HRP, and 
not endogenous peroxidase.

Second, you appear to be missing two other key blocking steps. 
One is a normal serum block. This is a step that can be inserted 
either before or after the primary antibody, and is usually a normal 
serum blocking solution made of the same species as your 
secondary Ab (e.g. if using Goat anti-mouse HRP, use a 1% 
solution of normal goat serum) to block nonspecific binding sites in 
the tissue. The other missing blocking step, is a step to block 
endogenous biotin in the tissue. In mammals at least, the gut can 
be rich in biotin. So the Avidin label may well be sticking to 
caterpillar biotin rather than your reagent biotin. Someone else on 
this list can give you a homemade recipe for biotin block. I just buy 
a biotin blocking kit from Dako.
Cheers! Greg

Date sent:      	Thu, 19 Apr 2001 16:06:18 +0100 (BST)
From:           	"J.L. Turnbull" <>
Subject:        	IHC help please - brown everywhere
Forwarded to:

> 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.
> Questions:
> 1) the protcol said that the iodine step was to remove the sublimate, what
> 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
> damage?
> 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
> difference.
> 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)
> Jenny

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