1. 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 4ºC.
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.
rat anti-mouse CD44 (IM7) mAb, BD Biosciences #553130 or
mouse anti-human CD44v3 mAb, R&D systems BBA11
horseradish peroxidase (HRP)-conjugated rabbit anti-rat Ig, Dako #PO450 (alternative HRP-rabbit anti-rat IgG, Sigma #A5795)
or HRP-rabbit anti-mouse IgG, Invitrogen #31450 (for use with CD44v3 mAb as primary ab)
Purified NA/LE Rat IgG2bκ , BD Biosciences #555845 or
Purified Mouse IgG2bk, BD Biosciences#557351
Hydrogen peroxide (30% w/w), Chem-Supply Pty Ltd (Australia) #HA154-500M,
Methanol, Merck # CAS-No. 67-56-1
3Amino-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