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
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