[Histonet] Secondary antibody causing nuclear staining?

Tue Jul 24 08:17:59 CDT 2012

These samples were all human but I have seen it in Mouse mammary gland as
well but those nuclei were lighter.
Eva

On Tue, Jul 24, 2012 at 8:51 AM, James Burchette Jr. <
james.burchette <@t> duke.edu> wrote:

> Thanks Eva. I don't know why you are having the nuclear staining problem.
> Your retrieval process isn't overly aggressive. I've used Vectors secondary
> and ABC reagents forever and have never had the issue you are describing.
> Human tissue?
>
> 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 8:41 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: Re: [Histonet] Secondary antibody causing nuclear staining?
>
> 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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> > >
> > >
> > >
> >
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