RE: Negative slide has specific staining

From:Todd Sherman

Hello Jennifer,

Greg and Rob have offered some good suggestions, but it would appear that you are still having difficulty detecting the 
DNP in frozen/acetone-fixed mouse lung.  I'm not sure what you mean when you say "my positive is coming up
red all over" but I'll not address that here.  What got my attention was your most recent comment that the antibody was 
"scavenged" (my term) from a WB kit.  That might be another source of problems.

The WB assay Rb x Ms-DNP Ab was designed/optimized to detect a sequence on the linearized protein on a gel 
electrophoresis apparatus; consequently, tertiary protein structure, and its associated, potentially three-dimensional 
binding region, has been disrupted to produce an accessible protein-binding site.  The binding region against which the 
assay Ab is directed could be proximal, terminal, hydrophilic, or hydrophobic (you'll have to check the manufacturer 
spec sheets for that).  To generalize greatly, if the binding region is located on a "free" end, i.e. proximally or terminally 
and hydrophilic, then the likelihood of successful specific binding using WB kit Ab in IHC is increased; conversely, if the 
binding region is hydrophobic and buried within the tertiary protein structure, then the likelihood of successful specific 
binding using WB kit Ab in IHC is decreased.

Antibodies designed/optimized for IHC usually take into account the tertiary structure of the binding site of interest.  The 
Ab is often directed against a site composed of several domains that are not in a strict linear sequence.  If the IHC-
optimized Ab is used in IHC, specificity can be maintained.  However, take this Ab and use it for a WB assay and the 
Ab might not bind to the protein of interest - it is "out of its element" since the tertiary structure disruption (from WB-gel 
denaturing agents) would be too significant.

What is the upshot of this?  Using an antibody from a kit optimized for WB assays may be the problem.  Manufacturers 
that have worked with specific proteins for extended periods of time often produce several Abs directed against the 
same protein and specific for different applications - a WB assay linear-sequence directed Ab and an IHC tertiary-
structure directed Ab.

My final $0.02> I hate to present potentially bad news but you should consider this point.  The antibody from your kit 
might not be detecting anything specific at all, even in the negative control.  Epithelial cells (alveolar epithelial type II 
pneumocytes) can be quite "sticky" since they synthesize pulmonary surfactant and aid in local immune defense 
mechanisms, so the detection you have previously visualized in the negative control could be non-specific.  [Anecdote 
from personal experience: we performed IHC studies to detect estrogen receptor in rat/sheep lung epithlium and found 
exceptional, voluminous, and seemingly specific staining in lung epithelium but we ended up concluding that it was non-
specific.]  Without going into all of the details of your study, I have found that the most important aspect of successful 
IHC is tissue fixation, antigen retrieval, and primary antibody.  Since the tissues were fixed in a routine manner and HIER 
was not needed, that leaves primary antibody as the most likely source of the problem in my mind.

Hope this helps,
Todd Sherman

10/12/2001 7:47:24 AM, "Berger, Jennifer"  wrote:

>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 []
>> Sent:	Thursday, October 11, 2001 5:21 PM
>> To:	'Berger, Jennifer '; ''' '
>> 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: ''
>> 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:
>> > 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
>> 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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