Thank you for the clarification and the answers of all others On Wed, Apr 13, 2022, 2:05 AM Tony Henwood (SCHN) < tony.henw...@health.nsw.gov.au> wrote:
> "Bond wash" is the propriety buffer used in the Bond Immunostainer. > > *Regards* > *Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)* > *Principal Scientist, the Children’s Hospital at Westmead* > *Adjunct Fellow, School of Medicine, University of Western Sydney* > *Tel: 612 9845 3306* > *Fax: 612 9845 3318* > *Pathology Department* > *the** children's* *hospital* *at westmead* > *Cnr Hawkesbury Road and Hainsworth Street, Westmead* > *Locked Bag 4001, Westmead NSW 2145, AUSTRALIA* > ------------------------------ > *From:* jayalakshmy p.s <psjayalaks...@gmail.com> > *Sent:* 12 April 2022 22:01 > *To:* Tony Henwood (SCHN) <tony.henw...@health.nsw.gov.au> > *Cc:* histonet@lists.utsouthwestern.edu <histonet@lists.utsouthwestern.edu > > > *Subject:* Re: [Histonet] Clarification about Immunohistochemistry > > Thank you so much Susan and Tony. Let me prepare the reagents &try. > To Susan- can you please elaborate on the steps of the giemsa method. > To Tony Henwood - please tell what is meant by "bond washing" > Thanks > Dr. Jayalakshmy > > On Tue, Apr 12, 2022, 9:58 AM Tony Henwood (SCHN) < > tony.henw...@health.nsw.gov.au> wrote: > > Hi there, > > I have had good results with Giemsa-like counterstaining (Stefanović et > al 2013, Ravishankar et al 2016) : > > Azure blue, when substituted for hematoxylin as a counterstain in > immunostain preparation, has been used to help differentiate melanocytes > from melanophages. Azure blue preferentially stains cytoplasmic melanin > granules blue-green, whereas melanocytes are highlighted by brown DAB > chromogen. Melanophages, which contain melanin and lack melanocytic > determinants, appear clear with blue-green granules in the cytoplasm > (Hillesheim et al 2011). > > After the slides were bond washed for 4min and rinsed in distilled water, > they were stained with a mixture of the following solution: 100 mg of Azure > blue (Sigma) in 4 ml of distilled water. Solution 2 was prepared as > follows: 0.6ml of 0.1M sodium acetate and 3.4 ml 0.1M acetic acid were > added to 27ml distilled water. Both solutions 1 and 2 were combined and 5ml > of acetone was added. The slides were incubated for 60 min at room > temperature, differentiated in 95% ethanol and dehydrated in several > changes of absolute ethanol, followed by clearing in xylene with subsequent > mounting (Kamino & Tarn 1991, Hillesheim et al 2011). > > An alternate method is to counterstain the immunohistochemical reaction > with a methylene blue solution (method courtesy of Dr Vince Munro, St > Vincents Hospital in Sydney): > > Staining Solution > 2.38gm Sodium acetate > 4.7ml Acetic acid > 5gm Methylene Blue > Make up to 1 litre with distilled water > > Procedure > 1. After immunostaining, wash slides in tap water > 2. Stain in Methylene Blue solution for 2 minutes > 3. Wash well in water > 4. Counterstain in Haematoxylin, wash well & blue as usual. > 5. Dehydrate, clear and mount > > Result: Melanin should stain green-blue. > > Hillesheim, P. B., Slone, S., Kelley, D., Malone, J., & Bahrami, S. > (2011). An immunohistochemical comparison between MiTF and MART‐1 with > Azure blue counterstaining in the setting of solar lentigo and melanoma in > situ. Journal of cutaneous pathology, 38(7), 565-569 > > Kamino H, Tarn ST (1991) Immunoperoxidase technique modified by > counterstain with azure B as a diagnostic aid in evaluating heavily > pigmented melanocytic neoplasms. Journal of cutaneous pathology, > 18(6):436-439. > > Ravishankar, S., Nagarajan, P., Curry, J. L., Tetzlaff, M. T., Ivan, D., > Torres-Cabala, C. A., ... & Prieto, V. G. (2016). Giemsa is the optimal > counterstain for immunohistochemical detection of BRAF V600E mutation > status in pigmented melanomas. Journal of cutaneous pathology, 43(8), > 722-724. > > Stefanović, D., Stefanović, M., & Nikin, Z. (2013). Romanowsky-Giemsa as a > counterstain for immunohistochemistry: optimizing a traditional reagent. > Biotechnic & Histochemistry, 88(6), 329-335. > > > Regards > Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) > Principal Scientist, the Children’s Hospital at Westmead > Adjunct Fellow, School of Medicine, University of Western Sydney > Tel: 612 9845 3306 > Fax: 612 9845 3318 > Pathology Department > the children's hospital at westmead > Cnr Hawkesbury Road and Hainsworth Street, Westmead > Locked Bag 4001, Westmead NSW 2145, AUSTRALIA > > > > > -----Original Message----- > From: jayalakshmy p.s via Histonet [mailto: > histonet@lists.utsouthwestern.edu] > Sent: Tuesday, 12 April 2022 1:59 PM > To: histonet@lists.utsouthwestern.edu > Subject: [Histonet] Clarification about Immunohistochemistry > > Hai all Histonetters > Please anybody clarify my this doubt if possible. > When doing Immunohistochemistry for confirmation of Melanoma with DAB > chromogen(brown color)the interpretation is not possible because of > obscuring by the dense melanin pigment. We dont have any other color > chromogen. I tried ihc after bleaching but the section gets detached even > from charged slides. Is there any other effective way to do this? > Thanks in advance > Regards > Dr. P S Jayalakshmy > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > This message is intended for the addressee named and may contain > confidential information. If you are not the intended recipient, please > delete it and notify the sender. > > Views expressed in this message are those of the individual sender, and > are not necessarily the views of NSW Health or any of its entities. > > > This message is intended for the addressee named and may contain > confidential information. 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