Hi
Right now, the positive control is to first treat 1 slide in H2O2.
Theoretically that should form a bunch of carbonyls-my positive is coming up
red all over, so it appears to be working.

My procedure in a nutshell is this:
Mouse lungs are snap frozen in liquid nitrogen.
I have our histo person cut frozen sections at 5um.
The slides are fixed in acetone, and stored at -80
Slides are then rehydrated in a PBS-Brij solution for 15 minutes.
Positive controls are placed in 1:50 H2O2 for 2 min
Slides are Derivitized using 10mM DNPH in 10%trifluoroacetic acid for 1 hour
3% H2O2 in Methanol for endogenous peroxidase for 20min
Block with 10% NGS for 30 min
Rabbit Anti-DNP antibody or rabbit IgG(neg) for 2hrs at 37C (diluent is
1%NGS in PBS-Brij )
biotinylated Goat AntiRabbit for 30 min (diluent is 1%NGS in PBS-Brij )
Vector NovaRed Substrate for 15 min
Counterstain with 1:3 Hematoxylin
Dehydrate and coverslip

We're actually getting our primary from a kit.  The kit is originally to be
used in a western blot assay.  We developed a way to use the kit as an ELISA
for lavage samples.  Now I'm trying to find a way to get it to work in
tissue samples.




> -----Original Message-----
> From:	Robert Geske [SMTP:RGeske@lexgen.com]
> Sent:	Thursday, October 11, 2001 5:21 PM
> To:	'Berger, Jennifer '; ''histonet@pathology.swmed.edu' '
> Subject:	RE: Negative slide has specific staining
> 
> Hi Jennifer,
> 
> can you tell us what your positive control is? not the experimental group
> that you expect to be positive but a historical known positive.
> 
> i would start with that known positive and then titer my primary down
> until
> it is extinguished and then run the non-immune isotype antibody at
> concentrations at the lower end of the series.  you might want to adsorb
> your antibody and IgG against normal mouse serum.  i usually use 2% normal
> mouse serum in my diluent and rock the antibody at room temp for an hour
> or
> hour and a half before application to sections. i'm sure you've considered
> this, but are you sure of the concentration calculations  --- you
> mentioned
> you repeated the experiment, but did you do that with the same batch of
> reagents that you had made from the previous problematic run or did you
> make
> fresh reagents? do you have any other istotype and species matched
> antibodies that you could apply to your sections (at the same titer you
> use
> this antibody at) where you know the immunoreactive sites should  and
> should
> not be be in the lung and see if it too sticks non specifically to what
> you
> are calling non specific staining with the IgG. 
> 
> just out of curiosity, since you are looking for protein carbonyls, what
> if
> anything are you fixing your frozen sections with? have you tried treating
> your sections (alcohol fixed, no aldehyde fixative)with Schiff reagent
> without oxidation (periodic acid) to see if there is a demonstrable,
> qualitative increase in tissue carbonyls in your experimental groups --
> you
> could do this with and without prior treatment with a reducing agent.
> 
> rob
> 
> -----Original Message-----
> From: Berger, Jennifer
> To: 'histonet@pathology.swmed.edu'
> Sent: 10/11/01 8:27 AM
> Subject: Re: Negative slide has specific staining
> 
> 
> No mix up.  They're frozen tissue.  Slides are all of the same
> animal-smoke
> exposed mice.  The slides are all treated exactly the same until the
> antibody is applied.  All slides except the neg control get primary, the
> neg
> gets IgG-My first thought was that I mixed up antibody and IgG, so I
> reran
> it with the same results.
> Jennifer
> > -----Original Message-----
> > From:	Greg Dobbin [SMTP:dobbin@Upei.CA]
> > Sent:	Thursday, October 11, 2001 5:29 AM
> > To:	Berger, Jennifer
> > Subject:	Re: Negative slide has specific staining
> > 
> > Hi Jennifer,
> > What about a mix up prior to cutting? As in mislabelled fixation jars,
> 
> > or a mix up when trimming. Sounds like a mix up to me!?
> > Greg
> > 
> > Date sent:      	Wed, 10 Oct 2001 18:55:00 -0600
> > From:           	"Berger, Jennifer" 
> > Subject:        	Negative slide has specific staining
> > Forwarded to:   	DOBBIN@acad1.cs.upei.ca
> > To:             	"'histonet@pathology.swmed.edu'"
> > 
> > 
> > > 
> > > Hi everyone
> > > I have a strange problem here.
> > > I'm doing some IHC for protein carbonyls on mouse lungs exposed to
> > cigarette
> > > smoke.
> > > 
> > > I'm blocking with goat serum.
> > > My primary antibody is Rabbit Anti-DNP
> > > My negative control is a slide that gets rabbit IgG instead of the
> > primary.
> > > My secondary in biotinylated Goat Anti-Rabbit.
> > > 
> > > Okay, my problem is that the ONLY slide that shows any of what
> appears
> > to be
> > > specific staining (type 2 cells) is the negative slide (IgG instead
> of
> > > antibody).
> > > I've rerun it with the same results, so it's not that I got the
> slides
> > mixed
> > > up.
> > > Any ideas?
> > > 
> > > Thanks
> > > Jennifer
> > > 
> > > 
> > 
> > 
> > >
> > >
> > >
> > >
> > >
> > >
> > Greg Dobbin
> > Pathology Lab
> > Atlantic Veterinary College, U.P.E.I.
> > 550 University Ave.
> > Charlottetown, P.E.I.
> > Canada,  C1A 4P3
> > Phone: (902)566-0744
> > Fax: (902)566-0851
> 
> 
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