Irrelevant control antibodies

Immunohistochemistry was performed on OCT cryosections or paraffin-embedded sections of 23 representative aortic specimens using a polyclonal rabbit anti-human vWf (Dako, #A0082) as primary antibody diluted to 10 μg/ml in TBS/TC (Tris-Buffered Saline—0.2% Tween20–0.6% casein, pH6.0) and a peroxidase LSAB-DAKO kit (Dako) for detection. The binding reaction was detected by DAB (3,3'-diaminobenzidine). Slides were then counterstained with Nuclear red (nucleus)/Alcian blue (proteoglycans). Irrelevant control antibodies (Dako) were applied at the same concentration in order to assess non-specific staining.

Human tissue samples were fixed in 3.7% paraformaldehyde for 2 days and decalcified in a solution of 14% ethylenediaminetetraacetic acid (EDTA) in distilled water, pH 7.4 for 4 to 6 weeks at 4 °C. Samples were embedded in paraffin wax and serially sectioned (5 µm) (n = 8–15). OA cartilage damage was evaluated on Safranin-O Fast Green stained tissue slices by a Mankin’s score (range 0–14) [59 (link)].
Immunohistochemistry was performed with mouse monoclonal antibody to Grem-1 (Abcam, dilution 1:50), BMP-4 (Abcam, dilution 1:50), VEGFR-2 (Abcam, dilution 1:50), BMPR-1a (Abcam, dilution 1:50), BMPR-1b (Abcam, dilution 1:50), BMPR-2 (Abcam, dilution 1:50), as primary antibodies. Enzyme-induced antigen retrieval was performed as follows: 0.2 mg/mL hyaluronidase in PBS, pH 5.5, for 10 min at 37 °C and then 0.1 mg/mL pronase in PBS, pH 7.4, for 20 min at 37 °C. The R.T.U Vectastain kit (Vector) was used for detection, followed by counterstaining with Mayer’s hematoxylin. Irrelevant control antibodies (Dako) were incubated at the same concentration to assess non-specific staining. Preparations were mounted in Eukitt medium.