H3506

Collagen 2 immunohistochemistry was performed as previously described58 (link) using mouse monoclonal anti-collagen 2 antibody (II-II6B3; DSHB Iowa) with Dako EnVision detection kit. In brief, slides were treated with 0.1% pronase (P-8811; Sigma) in PBS for 20 minutes at room temperature, followed by 2.5% hyaluronidase (H-3506, Sigma) in PBS for 30 minutes at 37 °C. Endogenous peroxidase activity was quenched with 3% H₂O₂ for 10 minutes at room temperature. Nonspecific binding sites were blocked with 10% goat serum in PBS for 60 minutes at room temperature. Sections were then incubated with primary antibody against collagen type 2 diluted 1:50 in PBS overnight at 4 °C. The slides were then washed in PBS, and incubated with the secondary antibody (Dako Real Envision/HRP, K5007) for 30 min at room temperature. DAB (Dako Real DAB+Chromogen, K5007) was applied for about 2 min and then removed by rinsing with distilled water. Slides were mounted with Biomount Aqua (Biognost, Croatia) and covered. Exclusion of primary antibody resulted in no staining.

Spermatogenic cells were purified using a method previously described^{46 (link)}. Briefly, testes from adult mice were removed and decapsulated. The seminiferous tubules were tore into small pieces and incubated in 10 ml DMEM (12100-046, Gibco) containing 1 mg/ml collagenase (C5138, Sigma) and 1 mg/ml hyaluronidase (H3506, Sigma) at 37 °C for 5 min with gentle shaking. After pipetting, the dispersed seminiferous tubules and cells were incubated at 37 °C for 5 min with gentle shaking. Then the cells were collected by centrifugation at 200 × g for 5 min at 4 °C, washed once with PBS, resuspended in 10 ml PBS containing 0.25% Trypsin and 1 mg/ml DNase I, and incubated at 37 °C for 5 min with gentle shaking. Cells were collected by centrifugation at 600 × g for 5 min and washed with 10 ml DMEM containing 0.5% BSA. After filtration through a 40 μm Nylon Cell Strainer, the cells were separated by 3 h of velocity sedimentation at unit gravity, using a 2–4% BSA gradient in DMEM. The cell fractions were bottom-loaded in a volume of 300 ml. The cell type and purity in each fraction were assessed using light microscope based on their diameters and morphological characteristics. Only fractions with the expected cell type and purity (≥90%) were pooled together.

Rabbits were euthanized under American Veterinary Medical Association guidelines and knees were isolated within 2 h of euthanasia. This study uses rabbits obtained from University of Texas Health Sciences (Dr. Steven Mills). Baylor College of Medicine did not require the study to be reviewed or approved by an ethics committee because we received dead animals. The articular knee joints were dissected under sterile conditions, and articular cartilage was isolated from both the femoral condyle and the tibial plateau. Isolated cartilage was diced into <1 mm (Mueller and Tuan, 2011 (link)) pieces before sequential digest, first in hyaluronidase for 30 min (660 Units/ml Sigma, H3506; in DMEM/F12 with pen/strep/amphotericin B, 30 mL), followed by collagenase type II for ∼16 h at 37°C (583 Units/ml Worthington Biochemical Corp.; in DMEM/F12 with 10% FBS, 1% pen/strep/fungizone, 30 mL). The digest was then filtered through a 70 μm cell strainer, washed with DMEM/F12, and resuspended in growth media (DMEM/F12 supplemented with 10% FBS, 1% pen/strep). Cells were subsequently infected as described below or cryopreserved (95% FBS, 5% DMSO).

To qualitatively evaluate the effectiveness of enzymatic degradations, 3 μm sections were prepared for Picrosirius red and Safranin O [21 (link)] stains to determine the collagen and the GAG distributions, respectively. The Picrosirius Red -staining protocol used in our study was modified from the standard protocol [22 (link)] by adding the purification step to demonstrate collagen fibre patterns. This was conducted by 18 h immersion in 0.5% papain (Acros Organics, 416761000) in 0.05 M PBS (pH 4.4, 37°C) and 1000 U/ml hyaluronidase (Sigma-Aldrich, H3506) in 0.1 M PBS (pH 6.9, 37°C) digestions before the staining with 0.1% Direct red 80 in saturated picric acid (Sigma-Aldrich, 365548). Polarized light microscopy (PLM) was used to measure the retardance caused by picrosirius red stain in the sections to reveal alterations in the collagen content. The sections were imaged with Abrio polarized light imaging system (CRi, Inc., Woburn, MA, USA) mounted on a light microscope (Nikon Diaphot TMD, Nikon, Inc., Shinagawa, Tokyo, Japan).