Immunocytochemistry/Immunofluorescence analysis of Phospho-Caveolin-2 pTyr19 (green) showing staining in the cytoplasm and nucleus of HUVEC cells treated with 100µM pervanadate (left) and untreated HUVEC cells (right). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with ab3417 in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

IHC image of ab3417 staining in human normal cervix formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab3417, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

ab3417 at a 1/250 dilution staining Caveolin-2 in rat fibroblasts by Immunocytochemistry/ Immunofluorescence.Cells were fixed in paraformaldehyde, permeabilized with Triton X-100 and blocked using 1% BSA. The secondary used was a TRITC conjugated anti-rabbit at a 1/100 dilution.(a) Control cells untreated cells (b) 100nM Insulin for 10 min (c) 10uM U0126 for 2 hr and 100nM Insulin for 10 min (d) 100 nM Wortmannin for 1hr and 100nM Insulin for 10 min