Alexa fluor conjugated goat anti rabbit igg

Cardiac myocytes were fixed with 2% paraformaldehyde, blocked with 1% bovine serum albumin and 0.1% Triton X-100 in 10% PBS. The cells were incubated with anti-RyR monoclonal antibody (C3-33, Thermo scientific) for 24 hours at 4 °C, washed with PBS and then incubated with Alexa Fluor-conjugated goat anti-rabbit IgG (Life Technologies) for 2 hours at room temperature. The immuno-staining was visualized using Leica SP5 confocal microscope at 488 nm (FKBP-YCaMP and FKBP-GCaMP6) and 540 nm (Alexa-Fluor) excitation and 500-550 and >560 nm emission, respectively.

Transverse frozen tissue sections (20μm) were rinsed, blocked, and incubated with polyclonal rabbit anti-GFAP (1:500, Dako, Glostrup, Denmark), or monoclonal rabbit anti-Claudin-1 (1:50, Cell Signaling Technology, Danvers, MA) overnight in 4°C. Sections were washed, incubated with AlexaFluor-conjugated goat anti-rabbit IgG (1:1000, Life Technologies, Carlsbad, CA) and counter-stained with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (1:50,000, Sigma-Aldrich, St. Louis, MO). Images were acquired with 20× and 63× objectives by confocal microscopy (Leica Microsystems, Wetzlar, Germany) with acquisition parameters kept constant. Analyses were performed on images captured with the 20× objective using ImageJ software (https://imagej.nih.gov/ij/). Mucosal regions indicated by apical epithelial cells and the submucosal border were outlined using the freehand tool. Parameters for image adjustments were set based on threshold signals and kept constant for all images. Mean gray value and area of outlined regions were measured, and integrated density of immunofluorescent signal was calculated.