RE: Negative slide has specific staining

From:Robert Geske

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.


-----Original Message-----
From: Berger, Jennifer
To: ''
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
exposed mice.  The slides are all treated exactly the same until the
antibody is applied.  All slides except the neg control get primary, the
gets IgG-My first thought was that I mixed up antibody and IgG, so I
it with the same results.
> -----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:
> To:             	"''"
> > 
> > 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
> to be
> > specific staining (type 2 cells) is the negative slide (IgG instead
> > antibody).
> > I've rerun it with the same results, so it's not that I got the
> 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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