Mouse anti α actinin primary antibody

Example 7

To confirm ChR2 expression (in the primary rat CMs, in the CM/ChR2-HEK co-cultures, and the iPS-CMs) and to confirm myocyte-like properties of IPS-CMs, antibody staining and confocal imaging was performed (see FIGS. 5A, 5B, and 16, panels “a” and “c”), using the OLYMPUS™ FLUOVIEW™ FV1000 confocal system. Samples were fixed in 3.7% formaldehyde after performing functional experiments. Prior to antibody labelling, cell membrane permeabilization was performed by incubating samples in 0.02% TRITON™ X-100 for five minutes. Cells were labelled with mouse anti-α-actinin primary antibody (SIGMA ALDRICH®, A-7811) at 1:600 and ALEXA FLUOR® 647 goat anti-mouse IgG secondary antibody (INVITROGEN™, A-21235) at 1:1000. All antibodies were diluted using 1% bovine serum albumin (AMERSHAM™ PLC, Amersham, UK). 1% FBS was used as a blocking agent. After antibody staining, cell nuclei were stained with 1 μg/mL DAPI with 10 minute incubation in PBS. Imaging was done using the OLYMPUS™ FLUOVIEW™ FV1000 confocal system with acquisition rate at 4 μs/pixel. Gain was kept constant for control and test groups to normalize and exclude autofluorescence contributions.