Re: Immunohistochemistry Puzzler
| From: | Geoff McAuliffe <mcauliff@UMDNJ.EDU> |
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Elizaha@aol.com wrote:
> Hi,
> There is an odd problem with an antibody we are using in our lab.
> It is
> a rabbit polyclonal, we use a streptavidin/biotin (commercial) kit,
> and
> chromagen is DAB. Tissue: chondrocytes/cell culture fixed in
> paraformaldehyde
> and embedded in paraffin..
> Problem: When we stain cells with it we get little to no label, if we
> add a
> hydrogen peroxide blocking step before the primary antibody we get
> heavy
> labeling of cells that looks specific/intracellular. The antibody is
> made "in
> house" but is not against hydrogen peroxide? Has anyone seen any
> reaction
> like this. Is this weird or what?
>
> Thanks again,
> Elizabeth
Dear Elizabeth:
My guess, and I do mean guess, is that the peroxide is "etching" or
"opening up" the antigenic sites to make them more available for
binding. Hydrogen peroxide treatment can dramatically speed up the
staining of EM grids by etching the surface of the epoxy sections. What
happens when you use the peroxide block but omit the primary antibody?
Are you getting all of the wax out of the sections?
Geoff
--
**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029
mcauliff@umdnj.edu
**********************************************
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Elizaha@aol.com wrote:
<blockquote TYPE=CITE><font face="arial,helvetica"><font size=-1>Hi,</font></font>
<br><font face="arial,helvetica"><font size=-1> There
is an odd problem with an antibody we are using in our lab. It is</font></font>
<br><font face="arial,helvetica"><font size=-1>a rabbit polyclonal, we
use a streptavidin/biotin (commercial) kit, and</font></font>
<br><font face="arial,helvetica"><font size=-1>chromagen is DAB. Tissue:
chondrocytes/cell culture fixed in paraformaldehyde</font></font>
<br><font face="arial,helvetica"><font size=-1>and embedded in paraffin..</font></font>
<br><font face="arial,helvetica"><font size=-1>Problem: When we stain
cells with it we get little to no label, if we add a</font></font>
<br><font face="arial,helvetica"><font size=-1>hydrogen peroxide blocking
step before the primary antibody we get heavy</font></font>
<br><font face="arial,helvetica"><font size=-1>labeling of cells that looks
specific/intracellular. The antibody is made "in</font></font>
<br><font face="arial,helvetica"><font size=-1>house" but is not against
hydrogen peroxide? Has anyone seen any reaction</font></font>
<br><font face="arial,helvetica"><font size=-1>like this. Is this weird
or what?</font></font>
<p><font face="arial,helvetica"><font size=-1>Thanks again,</font></font>
<br><font face="arial,helvetica"><font size=-1>Elizabeth</font></font></blockquote>
Dear Elizabeth:
<p> My guess, and I do mean guess, is that the peroxide
is "etching" or "opening up" the antigenic sites to make them more available
for binding. Hydrogen peroxide treatment can dramatically speed up the
staining of EM grids by etching the surface of the epoxy sections. What
happens when you use the peroxide block but omit the primary antibody?
<br> Are you getting all of the wax out of the sections?
<p>Geoff
<br>--
<br>**********************************************
<br>Geoff McAuliffe, Ph.D.
<br>Neuroscience and Cell Biology
<br>Robert Wood Johnson Medical School
<br>675 Hoes Lane, Piscataway, NJ 08854
<br>voice: (732)-235-4583; fax: -4029
<br>mcauliff@umdnj.edu
<br>**********************************************
<br> </html>
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