Immunohistochemical techniques: the effect of melanin bleaching

Area of Science:

• Histopathology
• Biochemistry

Background:

• Immunohistochemical staining is a crucial technique in diagnostics.
• Bleaching steps are sometimes used to enhance staining specificity.
• The impact of bleaching on various antigens and staining steps is not fully understood.

Purpose of the Study:

• To determine which antigens withstand a specific bleaching protocol.
• To assess the resistance of immunohistochemical staining steps to bleaching.
• To evaluate the effect of bleaching on antibody specificity and chromagen stability.

Main Methods:

• A panel of 10 antigens was stained immunohistochemically.
• A bleaching step (potassium permanganate and oxalate) was introduced at various stages.
• Staining results were compared between bleached and unbleached samples.
• Antibody specificity and chromagen stability were also assessed.

Main Results:

• Four antigens (HMB-45, S-100, factor VIII-related antigen, collagen type IV) were unaffected by bleaching.
• Staining for CD-20 and CD-45 was enhanced, while hsp27 was reduced.
• Staining for CD-45Ro, CD-31, and Ulex/anti-ulex antibody was abolished.
• Antibody specificity was altered for UCHL-1 and L-26.
• No immunohistochemical staining steps after primary antibody application were resistant to bleaching.
• Chromagens (amino ethyl-carbazole, diaminobenzidine, chloro-naphthol) were sensitive to bleaching.

Conclusions:

• The resistance of antigens to bleaching varies significantly.
• Bleaching can alter antibody specificity and abolish staining for certain antigens.
• No step in the immunohistochemical procedure, including chromagen demonstration, is resistant to this bleaching protocol.