[Histonet] Clarification about Immunohistochemistry

[Tony Henwood (SCHN)]

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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