Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast carcinoma tissue sections labeling Calpastatin with Purified ab101684 at 1/100 dilution (0.59 µg/ml). Heat mediated antigen retrieval was performed Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunocytochemistry/ Immunofluorescence analysis of HaCaT (human skin keratinocyte) cells labeling Calpastatin with purified ab101684. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow cytometry analysis of HaCaT (human skin keratinocyte) labeling Calpastatin with purified ab101684 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as a secondary antibody. Isotype control - Rabbit monoclonal IgG (ab172730) (black). Unlableled control - Unlabelled cells (blue).

Immunohistochemical analysis of Calpastatin in formalin-fixed, paraffin-embedded Human breast carcinoma using ab101684 at 1/200 dilution.

Overlay histogram showing HeLa cells stained with ab101684 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab101684) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.