[Histonet] RE: Secondary antibody causing nuclear staining

[Kim Donadio]

Eva, 
        Have you gotten your problem resolved yet? I was curious as to what heat method you are using? Are you using a microwave? I'm sorry to be asking so many questions and not getting back to you quickly. I am thinking if you have irregular positive staining in the same Negative control tissue on different days that its a technique issue. Maybe the microwave is heating irregular? Ive also seen when using a microwave that I got better results when I would make sure they dont evaporate when transferring to next reagent and I circulated the buffer with a pipette in between the heating process.  
 
Now if your using a steamer < some people still do, dont laugh> then perhaps if its the second batch the retreival is hotter than for the first batch causing overstaining for that second batch?. 
 
Is there any possibliltiy at all some of the slides could be drying out during the staining process? I have seen nuclear artifact in some cases this happened to. 
 
Are you making up your DAB everyday, there are some DAB's that you have to make up and they get old quickly and dont stain as well later on. 
 
So many questions, sorry. I'd love to help you if you havnt figured this out yet. Please feel free to email me. 
 
Kim D
 
 


________________________________
From: Eva Permaul <eca9 <@t> georgetown.edu>
To: "Reynolds, Donna M" <dreynold <@t> mdanderson.org>; histonet <histonet <@t> lists.utsouthwestern.edu> 
Sent: Wednesday, July 25, 2012 10:59 AM
Subject: Re: [Histonet] RE: Secondary antibody causing nuclear staining

I do see positive nuclei in the NC. That is what I am asking about. I know
I could switch methods but my question is also why if it is happening is it
not as strong all the time? Why are the cells very light one day and dark
the next? What is causing them to stain? Just curious is all.
Eva

On Wed, Jul 25, 2012 at 10:04 AM, Reynolds,Donna M
<dreynold <@t> mdanderson.org>wrote:

>
> If you are running a negative control (no primary)with your ABC staining
> wouldn't you see the same nuclear labeling in the NC ? Thus alerting you to
> false staining and indicating that you should try a HRP conjugated
> secondary or use a polymer system.
> Good discussion thank Tony.
> Donna Reynolds HT(ASCP) Chief Histologist IHC Lab
>
> -----Original Message-----
> > 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
> >>
>
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