Staining method for type IV collagen alfa-1 (MAB 1), alfa-3 (MAB 3) and alfa-5 (MAB 5)

author MD Johan Mölne
category Immunohistochemistry
last updated at 2014-07-19 15:35:50

Staining method for type IV collagen alfa-1 (MAB 1), alfa-3 (MAB 3) and alfa-5 (MAB 5) using immunoperoxidase on formalin-fixed, paraffin-embedded tissue using Wieslab Alport’s Syndrome Immunohistochemical staining kit (Euro-Diagnostica)

1. Deparaffinize and rehydrate sections (3-4 μm thickness).
2. a. Antigen demasking (MAB 1,3, and 5)
Microwave boiling in citrate buffer, pH 2 at 750W for 8 min followed by 350W for 15 min. (Citrate buffer pH6 (Dako S2031) is adjusted with 1M HCl).
b. MAB 5 only Rinse in distilled water.
Incubate in Glycine/urea solution (provided in the Wieslab kit) for 10 min at 4-6°C Rinse in distilled tap water for 2 × 2 min.
3. Endogenous peroxidases are blocked using H2O2, 1.5% in TBS for 10 min.
4. Incubate with MAB 1 and MAB 3, dilution 1:50, MAB 5 1:100 for 20 min.
5. Incubate with secondary antibodies supplied with your detection kit. MAB 1 and MAB 3 with Anti-Mouse detection system.
Note! *MAB 5 with Anti-Rat detection system.

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