Prepare graded alcohols and xylene for deparaffinizing tissue sections: 2 x xylene (250 ml/slide container), 2 x absolute ethanol (250 ml/slide container), 1 x 90% ethanol (250 ml), 1 x 70% ethanol (250 ml)
2. Prepare acetate buffer components:
(i) 0.1N acetic acid: 290 μl glacial acetic acid in 50 ml deionized water
(ii) 0.1M sodium acetate: 410 mg anhydrous CH3COONa in 50 ml deionized water.
Prepare 0.1M acetate buffer (pH 5.2) by mixing 10.5 ml 0.1N acetic acid and 39.5 ml 0.1M sodium acetate.
3. Prepare stock solution of 3-amino-9-ethylcarbazole (AEC; chromogen, 8 mg/ml: 40 mg AEC in 5 ml N-N-dimethyl formamide; protect from light and refrigerate at 4°C.
4. Prepare M.O.M. diluent: 200 μl M.O.M. protein concentrate stock solution (M.O.M immunodetection kit) in 2.5 ml phosphate-buffered saline (PBS) for use either as a blocking step to minimize non-specific Ig binding or for diluting antibodies.
10E4 (mouse anti-human HS) mAb, Amsbio #370255-1
Mouse anti-mouse collagen type XVIII (Col18A1), Santa Cruz Biotechnol #1837-46
Horseradish peroxidase (HRP) -conjugated rabbit anti-mouse Ig, Dako #PO161 (alternatives: HRP-rabbit anti-mouse IgM, Thermo Fisher #31456 (for HS); HRP-rabbit anti- mouse IgG (H+L), Thermo Fisher#31450)
Mouse Igκ , BD Biosciences #550340
Mouse IgG2bκ , BD Biosciences #557351
Hydrogen peroxide (30% w/w), Chem-Supply Pty Ltd (Australia) #HA154-500M
Pronase, Calbiochem #537088
3-Amino-9-ethylcarbazole (AEC), Sigma-Aldrich #A5754
Animal free blocker, Vector Labs #SP-5030
Stock protein concentrate, M.O.M immunodetection kit, Vector Labs # PK-2200
N-N-dimethyl formamide, Sigma #D158550
Glycergel mounting medium, Dako #C0563