Staining Vinculin in mouse skeletal muscle tissue sections by Immunohistochemistry (Paraffin - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 3% BSA for 0.5 hour at room temperature; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody (1/25) for 1 hours at 37°C. A undiluted biotinylated goat polyvalent antibody was used as the secondary antibody.

Overlay histogram showing NIH/3T3 cells stained with Vinculin (green line). The cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with 90% methanol for 10 min. The cells were then icubated in 2% bovine serum albumin to block non-specific protein-protein interactions followed by the antibody (Vinculin, 1:25 dilution) for 60 min at 37ºC. The secondary antibody used was Goat-Anti-Rabbit IgG, DyLight 488 Conjugated Highly Cross-Adsorbed(OH191631) at 1/200 dilution for 40 min at 37ºC. Isotype control antibody (blue line) was rabbit IgG (1ug/1x10^6 cells) used under the same conditions. Acquisition of >10,000 events was performed.

All lanes: Anti-Vinculin at 1:2000 dilution Lane 1: human skeletal muscle lysate Lane 2: A431 whole cell lysate Lane 3: Hela whole cell lysate Lane 4: mouse kidney lysate Lane 5: PC-12 whole cell lysate Lane 6: HepG2 whole cell lysate Lysates/proteins at 20ug per lane. Secondary Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/10,000 dilution. Predicted band size: 117kD Blocking/Dilution buffer: 5% NFDM/TBST.