Alexa fluor 594 goat anti mouse

FDB fibers dissociated from control mice and mice < 1 hr after exercise were plated on glass bottom dishes and fixed in 4% paraformaldehyde for 20 min at room temperature. Following blocking for 1 hr at room temperature in 10% BSA in 1X PBS-T (PBS + 0.1% Triton X-100), fibers were co-labeled overnight at 4°C using primary antibodies for GFP (Rabbit polyclonal antibody, Thermo Fisher Scientific, Cat. # A11122, 1:3000) and either α-actinin (Mouse anti α-actinin, Sigma, Cat # A7811, 1:750) or the type1 ryanodine receptor (RYR1- Mouse anti RYR1 Cat # 34C, DHSB University of Iowa, 1:30). All primary antibody incubations were in 2% BSA in 1X PBS-T. Following three 10 min washes in PBS-T, samples were incubated with a 1:500 dilution of Goat anti Rabbit Alexa fluor 488 (Molecular Probes Cat # A11034) and Goat Anti Mouse Alexa fluor 594 (Molecular Probes Cat # R37121) for 1 hr at room temperature. Following three 10 min washes in 1X PBS-T, samples were imaged using an Olympus FV1000 laser scanning confocal microscope (Olympus Scientific Solutions, Wlatham, MA) and a 100X, UPlanSAPO NA 1.4 oil immersion objective. Alexa 488 and 594 were sequentially excited at 488 and 559 nm and detected at 515 and 617 nm respectively.

Immunostaining of the wing imaginal disc was performed according to standard protocol. Primary antibody anti-armadillo (AB_528089, Developmental studies hybridoma bank) and secondary antibody goat anti-mouse Alexa Fluor 594 (Molecular Probes, 1:500) were used.

Indirect immunofluorescence was performed on 10 μm control (GFP) Na_v1.5-WT or R104W-injected mouse ventricle cryosections fixed with paraformaldehyde for 15 min. Sections were washed twice for 5 min with phosphate buffer saline (PBS), blocked in PBS-5% bovine serum albumin (BSA) for 30 min at room temperature. Sections were then incubated overnight with primary antibodies at 4°C: the rabbit anti-GFP (1:2000, Torrey Pines Biolabs, United States) to detect exogenous hNa_v1.5-GFP, and mouse anti-α-actinin 2 (1:500, Sigma-Aldrich, United States). Heart sections were then washed twice with PBS and incubated 1 h with secondary antibodies: goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 594 (1:1000, Molecular Probes, Thermo Fisher Scientific, United States), and the nuclear dye DAPI (1:2000, Merck, Germany) diluted in the blocking buffer. Control experiments were performed by omitting primary antibodies.
Labeled ventricle sections were observed with a DeltaVision epifluorescent microscope (20× or 60×). Images were analyzed with DeltaVision imaging system (GE Healthcare, Seattle, WA, United States) equipped with 3D-deconvolution. For each sample, series of consecutive plans were acquired (sectioning step: 0.2 μm).

RLE cells were grown in glass coverslips and exposed to room air or hyperoxia for 18 h (for cleaved caspase‐3 or TUNEL staining) or 4 days (E‐cadherin staining). For E‐cadherin staining, coverslips were fixed in methanol at −20°C for 2 min, followed by 3 washes in PBS and blocking for 20 min in 5% BSA in PBS. Mouse anti‐E‐cadherin antibody (1:400) was used in 1% BSA in PBS for 1 h at room temperature, followed by 3 washes in PBS, secondary goat antimouse‐Alexafluor 594 (Molecular Probes) for 1 h at room temperature in the dark, three more washes in PBS and then coverslips were mounted onto slides using Prolong Gold antifade with DAPI. For cleaved caspase‐3 staining, a protocol provided by Cell Signaling was carefully followed, which included modifications in blocking solution and antibody dilution, and an overnight staining step with the rabbit monoclonal antibody against cleaved caspase‐3. Stained cells were observed under an Olympus BX60 fluorescence microscope, and pictures were taken.