Re: RE: IHC help please - brown everywhere

From:Jamie Erickson <JErickson@genetics.com>

HI Jenny,
                I have never worked with insects but here are my thoughts on IHC (immunohistochemistry) itself. First not all antibodies will work in fixed paraffin embedded  tissues for some reason some antibodies  just don't work and I've had to go to frozen blocks to get results. Second, when doing IHC and your not sure where the problem is you should eliminate step to rule them out. For example I would do one slide that has all my steps in it then the next slide I'd leave off the H202 step then the next slide leave the serum block off and the next slide leave off the primary antibody. By placing PBS (buffer) in those places in place of the reagent  you can look at all the slides and by the process of elimination you should be able to isolate the problem. IF you think it is your H2O2 try higher concentrations 0.5% 1 minute up to 3% for one minute, H2O2 is used to quench endogenous peroxidase in the tissue so that you only get a reaction with your peroxidase conjugated antibody not with cells in the tissue that contain peroxidase. Also there are many ways to antigen retrieve and in my hands if you over expose slides to enzymes or microwaving your whole section turns brown or the color of you chromogen. Here are some other enzymes to try: 0.05% pronase 10 minutes at 37, trypsin (Zymed) 15 minutes at 37, pepsin (Zymed) 10 minutes room temp, Proteinase K (25ug/ml) for 15 minutes at 37. AS far as chromogens go I use a DAB from Research Genetics (ready to use) no Filtering or a AEC from Zymed labs, they work great. I hope this helps in some way.


 

Jamie Erickson
Scientist II
Genetics Institute
1 Burtt Rd.
Andover, MA   01810
work : (978) 247-1348
FAX  : (978) 247-1389

>>> "Leek, Adrian" <ALeek@cytologix.com> 04/19 1:07 PM >>>
Jenny:
Unless you have a specific reason for it, I would also stop using the
hypertonic cacodylate.  Do you really want that much organic arsenic around?
Adrian Leek.


-----Original Message-----
From: J.L. Turnbull [mailto:jlt25@hermes.cam.ac.uk] 
Sent: Thursday, April 19, 2001 11:06 AM
To: histonet@pathology.swmed.edu 
Subject: IHC help please - brown everywhere



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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