[Histonet] Secondary antibody causing nuclear staining?

Tue Jul 24 18:23:58 CDT 2012

Hello Eva

I had a similar problem with an actin antibody many years ago.  We eventually after many trials decided that it was an artifact caused by the heat retrieval possibly in relation to the length of fixation time.  By playing with the dilution we eventually eliminated the problem by not retrieving that antibody at all and still got good antigenic staining.  While advent of  HIER has improved the staining of many antibodies it is not a necessity for all of them.

regards
Tony

Tony Reilly  B.App.Sc. , M.Sc.
Chief Scientist, Anatomical Pathology
Pathology Queensland-PA Laboratory 
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>>> Kim Donadio <one_angel_secret <@t> yahoo.com> 7/24/2012 10:18 pm >>>
Are you getting false positives and variations on the same control tissue for different days ? 

Sent from my iPhone

On Jul 24, 2012, at 8:13 AM, Eva Permaul <eca9 <@t> georgetown.edu> wrote:

> 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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