[Histonet] Clarification about Immunohistochemistry

[jayalakshmy p.s]

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.henwood at 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 at lists.utsouthwestern.edu]
> Sent: Tuesday, 12 April 2022 1:59 PM
> To: histonet at 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
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