Rabbit anti mouse secondary antibody

Cells were harvested by repeat pipetting and then centrifuged (14000 g, 1 min), washed in ice-cold PBS and re-pelleted as above. Cells were resuspended in ice-cold PBS supplemented with protease inhibitors (Complete EDTA-free Protease Inhibitor, Roche) and lysis was performed by 3×10 s bursts with a chilled probe sonicator (Microsonix). Protein concentration was determined using a modified Lowry protein assay (Biorad) and cell lysates (30 μg of protein) were resolved by SDS/PAGE [8%–10% (w/v) acrylamide]. Following transfer on to nitrocellulose membrane, ABCG2 was detected using BXP-21 (Merck Biosciences) at a 1:500–1:2000 dilution in PBS/5% (w/v) skimmed milk powder/0.1% (v/v) Tween, followed by a horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody (1:2000; DAKO). Chemiluminescence was detected using Supersignal West Pico (Pierce). Parallel gels were loaded and stained with Coomassie Brilliant Blue to ensure equal protein loading.

After 7 days in culture, the constructs were fixed in 10% formalin and embedded into paraffin. Sections with a thickness of 5 μm were cut. The presence of collagen type II was immunolocalized with mouse monoclonal antibody against collagen type II (Thermo Scientific, USA). After 30 min of room temperature incubation with primary antibody, sections were incubated with rabbit anti-mouse secondary antibody (DAKO, Denmark) for another 30 min. This is followed by positive staining visualization using 3,3′-Diaminobenzidine (DAB; DAKO) and counterstained nucleus in hemotoxylin. Microscopic images were photographed with a microscopy imaging system (Q550IW; Leica, Germany).

Cells were grown on Thermanox coverslips (Nalge Nunc International, Rochester, NY) and briefly fixed with 4% PFA/0.1 M cacodylate at a 1:1 ratio with culture medium. After 2 min, fixative was replaced with fresh 2% PFA/0.1 M cacodylate. Cells were blocked with 1% BSA, 0.1% Aurion BSA (Aurion, the Netherlands) before being incubated with mouse monoclonal antibodies against lumenal/extracellular antigens for 1 hr at room temperature, followed by incubation with a rabbit anti-mouse secondary antibody (Dako). The cells were then labeled with 10 nm protein-A gold (Utrecht university, the Netherlands), and re-fixed with 2% PFA, 2.5% glutaraldehyde / 0.1 M cacodylate, before being post-fixed with 1% osmium tetroxide and processed for conventional resin embedding as above. For cell surface cholesterol labeling, cells were fixed as above. Coverslips were then incubated with 1 μM TNM-BF for 30 min on ice, washed, and incubated with a rabbit polyclonal anti-BODIPY antibody followed by 10 nm protein-A gold as above.