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 <e...@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.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