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

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 Syndrome Immunohistochemicalstaining kit
(Euro-Diagnostika)
(This is a slight modification of an original method developed by Ole Nielsen, Odense University Hospital, Odense, Denmark)

1. Deparaffinize and rehydrate sections (3-4 mm thickness).

2. Antigen demasking – this has to be done in two different ways:

2a. MAB 1 and MAB 3: 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). Proceed to step 3.

2b. MAB 5: Microwave boiling in citrate buffer, pH 6 (Dako S2031) at 750W for 8 min followed by 350W for 5 min. Rinse in distilled water for 2 min. Digest the sections in Protease XXIV 0,05% (Sigma P8038) using Microwave oven at 37∞C, 5 min. Rinse in distilled water for 5 min. Incubate in Glycin/urea solution (provided in the Wieslab kit) for 10 min at 4-6∞C Rinse in distilled tap water for 2 × 2 min. Proceed to step 3.

3. Endogenous peroxidases are blocked using H2O2, 1,5% in TBS for 10 min. Rinse in tap water for 5 min, distilled water for 1 min. (We use EnVison FLEX Peroxidase-Blocking Reagent (SM801, Dako), 5 min)

4. Incubate with MAB 1 and MAB 3, dilution 1:50, and MAB 5 1:200 for 25 min.

5. Incubate in secondary antibodies followed by your detection kit.

We use Dako EnVision Flex Kit K8000 which is polymer based

6. EnVison Flex+ Mouse (LINKER) (SM804, Dako), 25 min.

7. EnVison FLEX/HRP (SM802, Dako), 20 min.

8. Substrate Working Solution (mix) (SM803, Dako), 5 min x 2.

9. EnVison FLEX Haematoxylin (SM806, Dako), 5 min

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