[Histonet] Secondary antibody causing nuclear staining?

Tue Jul 24 07:41:55 CDT 2012

We standard use a Citrate pH6. We do 20min at 98C followed by cooling in
the citrate for 20min.
Eva

On Tue, Jul 24, 2012 at 8:29 AM, James Burchette Jr. <
james.burchette <@t> duke.edu> wrote:

> What is your heat retrieval process?
>
> Jim Burchette, HT(ASCP) QIHC
> Histologist and Fly Fishing Bum
> Orlando, Florida
>
> ________________________________________
> From: histonet-bounces <@t> lists.utsouthwestern.edu [
> histonet-bounces <@t> lists.utsouthwestern.edu] on behalf of Eva Permaul [
> eca9 <@t> georgetown.edu]
> Sent: Tuesday, July 24, 2012 8:13 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: Re: [Histonet] Secondary antibody causing nuclear staining?
>
> I understand the point about the biotin and I should have said that when
> using the ABC method we have taken to always using an avidin/biotin
> blocking kit. We are using biotinylated secondary antibodies from Vector. I
> have seen the same problem occur in our anti-mouse, anti-rabbit and
> anti-goat. In my last run I had stomach fundus as well as skin melanoma,
> both had pos.nuclei in the negative (no primary). In another run I had
> colon ca and breast ca, the breast ca had fewer pos. nuclei than the colon
> ca but they were still there. Some days the positive nuclei are stronger in
> a sample that was just weakly positive before. Just want to understand what
> it is and what effects it.
> Thank you all for your ideas.
> Eva Permaul
> Georgetown University
>
> On Mon, Jul 23, 2012 at 7:16 PM, Tony Henwood (SCHN) <
> tony.henwood <@t> health.nsw.gov.au> wrote:
>
> > I should have added that this was from the workshop notes on a
> > Hypotheticals Workshop I ran last year at our Australian National
> Meeting.
> >
> > Regards
> > Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
> > Laboratory Manager & Senior Scientist
> > Tel: 612 9845 3306
> > Fax: 612 9845 3318
> > the children's hospital at westmead
> > Cnr Hawkesbury Road and Hainsworth Street, Westmead
> > Locked Bag 4001, Westmead NSW 2145, AUSTRALIA
> >
> >
> > -----Original Message-----
> > From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:
> > histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tony Henwood
> > (SCHN)
> > Sent: Tuesday, 24 July 2012 9:00 AM
> > To: 'Eva Permaul'; histonet <@t> lists.utsouthwestern.edu
> > Subject: RE: [Histonet] Secondary antibody causing nuclear staining?
> >
> > It is possible that this is due to "Biotin nuclei" where excess biotin is
> > found in the nuclei of some cells, see below:
> >
> > Optically clear nuclei have been reported in endometrial epithelium
> > associated with first and second trimester abortions (Sickel & di
> > Sant'Agnese 1994). Optically clear nuclei have also been found in
> different
> > types of tissues of diverse organs such as ovary, thyroid and lung
> > (Nakatani et al 1994, Mount & Cooper 2001). The optically clear nuclei
> > contain excess biotin.
> >
> > Endogenous biotin immunoreactivity is generally not visualized in
> formalin
> > fixed, paraffin-embedded tissues unless a heat-induced antigen retrieval
> > step has been introduced (Mount & Cooper 2001).
> >
> > In this placental section, optically clear nuclei (containing biotin)
> bind
> > to the streptavidin of the ABC technique giving a reaction similar to
> that
> > seen with CMV containing cells. If a polymer method (or even the original
> > Sternberger's PAP method) is used then this anomalous staining will
> > disappear, thus allowing confident demonstration of CMV infected nuclei.
> >
> > The false-positive staining pattern caused by endogenous biotin can be
> > cytoplasmic or nuclear. A report of positive immunoreactivity of
> > hepatocellular carcinomas for inhibin was later determined to be a
> > false-positive finding due to cytoplasmic endogenous biotin. Steroid cell
> > tumours of the ovary were found to demonstrate endogenous biotin
> > cytoplasmic staining in 36% of cases. Immunoreactivity for anti-Herpes
> > virus immunohistochemical staining in a series of endometria was also
> later
> > determined to be a false-positive result due to biotin. The prominent
> > intranuclear inclusions, resembling herpes virus cytopathic effect, were
> > caused by intranuclear biotin and not viral particles. Similar false
> > positive staining for CMV in products of conception has also been
> reported
> > (Mount & Cooper 2001).
> >
> > False-positive staining can be cytoplasmic or nuclear. When cytoplasmic,
> > the appearance of the false signal is that of a dull brown granular or
> > fluffy staining pattern. If this quality of staining is observed with
> > several different antibodies, endogenous staining by biotin should be
> > considered. When nuclear, a false-positive reaction may be associated
> with
> > optically clear nuclei identified on H&E stained sections. False-positive
> > staining due to endogenous biotin, however, does not occur in a cell
> > membrane pattern (Mount & Cooper 2001).
> >
> > Mount SL & Cooper K (2001) "Beware of biotin: a source of false-positive
> > immunohistochemistry" Current Diagnostic Pathology  7:161-167.
> > Nakatani et al (1994) Am J Surg Pathol 18(6):637-642.
> > Sickel & di Sant'Agnese (1994) Arch Pathol Lab Med 118:831-833
> >
> >
> > Regards
> > Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
> > Laboratory Manager & Senior Scientist
> > Tel: 612 9845 3306
> > Fax: 612 9845 3318
> > the children's hospital at westmead
> > Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001,
> > Westmead NSW 2145, AUSTRALIA
> >
> > -----Original Message-----
> > From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:
> > histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Eva Permaul
> > Sent: Monday, 23 July 2012 11:40 PM
> > To: histonet <@t> lists.utsouthwestern.edu
> > Subject: [Histonet] Secondary antibody causing nuclear staining?
> >
> > Hello,
> >
> > I have noticed that our biotinylated secondary antibodies on occasion
> > cause nuclear staining in some samples. Why is this? It is not every time
> > so I find it rather stange. Anyone know why this is happening and what I
> > can do to avoid it?
> >
> > Thank you for any suggestion,
> > Eva Permaul
> > Georgetown University
> > _______________________________________________
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> > Histonet <@t> lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> >
> >
> >
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