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.burche...@duke.edu> wrote: > What is your heat retrieval process? > > Jim Burchette, HT(ASCP) QIHC > Histologist and Fly Fishing Bum > Orlando, Florida > > ________________________________________ > From: histonet-boun...@lists.utsouthwestern.edu [ > histonet-boun...@lists.utsouthwestern.edu] on behalf of Eva Permaul [ > e...@georgetown.edu] > Sent: Tuesday, July 24, 2012 8:13 AM > To: histonet@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.henw...@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-boun...@lists.utsouthwestern.edu [mailto: > > histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tony Henwood > > (SCHN) > > Sent: Tuesday, 24 July 2012 9:00 AM > > To: 'Eva Permaul'; histonet@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-boun...@lists.utsouthwestern.edu [mailto: > > histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Eva Permaul > > Sent: Monday, 23 July 2012 11:40 PM > > To: histonet@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 > > _______________________________________________ > > Histonet mailing list > > Histonet@lists.utsouthwestern.edu > > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > > > > > > ********************************************************************************* > > This email and any files transmitted with it are confidential and > intended > > solely for the use of the individual or entity to whom they are > addressed. > > If you are not the intended recipient, please delete it and notify the > > sender. > > > > Views expressed in this message and any attachments are those of the > > individual sender, and are not necessarily the views of The Children's > > Hospital at Westmead > > > > This note also confirms that this email message has been virus scanned > and > > although no computer viruses were detected, The Childrens Hospital at > > Westmead accepts no liability for any consequential damage resulting from > > email containing computer viruses. > > > > > ********************************************************************************* > > > > _______________________________________________ > > Histonet mailing list > > Histonet@lists.utsouthwestern.edu > > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet