ICC/IF image of ab2868 stained PC12 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2868, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

ab2868 staining Ryanodine Receptor (red) in Mouse Skeletal muscle cells at day 10 of agrin-treated differentation by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.25% Triton X-100 in PBS and blocked with 10% serum for 45 minutes at 20°C. Samples were incubated with primary antibody (1/200 in PBS + 3% BSA) for 2 hours at 20°C. An Alexa Fluor^® 647-conjugated Donkey anti-mouse IgG polyclonal (1/500) was used as the secondary antibody. Nucleus stained blue.

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Immunohistochemistry was performed on biopsies of deparaffinized Human skeletal muscle tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing Ryanodine Receptor ab2868 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

ab2868 staining Ryanodine Receptor in Rat cardiomyocyte cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with methanol/acetone (1:1) and blocked with 3% BSA for 1 hour at 18°C. Samples were incubated with primary antibody (1/300 in PBS + 3% BSA) for 16 hours at 4°C. An Alexa Fluor^® 546-conjugated Goat anti-mouse IgG (H+L) polyclonal (1/300) was used as the secondary antibody.

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