Sp5 confocal microscopy

Immunofluorescence staining was performed as described previously. After fixation with 4% paraformaldehyde (PFA)/PBS, the hiPSC-CMs or MCOs were permeabilized and incubated overnight at 4ºC with primary antibodies against sarcomeric α-actinin (Sigma-Aldrich), diluted in 2% (v/v) HS/PBS. After 3 washes with PBS for 5 min, cells were incubated with 4',6-diamidino-2-phenylindole (DAPI Thermo Fisher Scientific) and AlexaFluor 555 anti-mouse (Thermo Fisher Scientific) secondary antibody for 1 h at room temperature. Dishes were mounted onto glass slides (Fisher Scientific) with a drop of ProLong™ Gold Antifade (Thermo Fisher Scientific). Fluorescent images were acquired with the SP5 confocal microscopy (Leica) using a 40 × magnification. Cell size was quantified blindly using the software Image J.

Adult brains were dissected and fixed with 4% formaldehyde in phosphate-buffered saline for 20 min whereas adult NMJ were fixed 10 min; in both cases, samples were washed 3 × 15 min with PBS+0.4% triton, blocked for 1 h with PBS+0.4% triton+ BSA 5%, incubated overnight with primary antibodies, washed 3 × 15 min, incubated with secondary antibodies for 2 h, and mounted in Vectashield mounting medium, with DAPI in the case of the brains. The primary antibodies used were anti-repo mouse (1/200; DSHB) to recognize glial nuclei, anti-bruchpilot-NC82-mouse (1/50; DSHB) to recognize the presynaptic protein bruchpilot, anti-HRP rabbit (1/400; Cell Signalling) to recognize membranes, anti-GFP rabbit (1:500; DSHB) and anti-Elav (1:100; DSHB) to recognize neuron nuclei. The secondary antibodies used were Alexa 488 or 647 (1/500; Life Technologies). Images were taken by a Leica SP5 confocal microscopy.

Cells were loaded with 10 µM cSNARF1 and 2.7 µM Hoechst 33,342 for 10 min at 37°. Cells were washed in neutral pH media and imaged in various pHe media (6.4–7.4) on Leica SP5 Confocal Microscopy × 40 objective. Cytoplasmic pH was measured by gating pixels according to a threshold level of Hoechst signal within cSNARF1-positive pixels. Fluorescence at 580 and 640 nm was averaged, background offset, and ratioed for each particle representing a cell.

All tissues were treated in simultaneously for each experiment. Adult brains were dissected and fixed with 4% formaldehyde in phosphate-buffered saline for 20 min, whereas adult NMJ were fixed for 10 min; in both cases, samples were washed 3 × 15 min with PBS + 0.4% triton, blocked for 1 h with PBS + 0.4% triton + BSA 5%, incubated overnight with primary antibodies, washed 3 × 15 min, incubated with secondary antibodies for 2 h and mounted in Vectashield mounting medium with DAPI in the case of the brains. The primary antibodies used were anti-Repo mouse (1/200; DSHB, Iowa City, IA, USA) to recognize glial nuclei, anti-Bruchpilot-nc82-mouse (1/50; DSHB, Iowa City, IA, USA) to recognize the presynaptic protein Bruchpilot, anti-HRP rabbit (1/400; Cell Signaling, Danvers, MA, USA) to recognize neuronal membranes, anti-GFP rabbit (1:500; DSHB, Iowa City, IA, USA) and anti-Myc guinea pig (1/100; DSHB, Iowa City, IA, USA) to recognize the nuclear protein Myc. The secondary antibodies used were anti-mouse, -rabbit or -guinea pig Alexa 488 or 647 (1/500; Life Technologies, Carlsbad, CA, USA). Images were taken by a Leica SP5 confocal microscopy applying same conditions for each experiment.

GV oocytes, MII oocytes, and preimplantation embryos were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. They were washed in PBS with 0.1% Triton three times and permeabilized with 0.25% Triton in PBS for 1 h at room temperature. The embryos were incubated with primary antibody overnight at 4 °C, after blocking in PBS with 0.1% Triton, 10% FBS and 5% BSA for 1 h at room temperature. Primary antibodies used in this study was α-SRSF3 (a kind gift from Dr Nielsen, 1:100), α-H3S10P (Abcam, 1:100), α-gamma-H2AX (Millipore, 1:100), α-H3K9me3 (Abcam, 1:100), α-H3K9me2 (Abcam, 1:100) and α-H3K9ac (Abcam, 1:100). The oocytes and embryos were then stained with secondary antibodies conjugated with Alexa Fluor 488 or 596 for 1 h at room temperature. DAPI was used for nuclear staining. The embryos were analyzed by Leica Sp5 confocal microscopy using a ×40 oil immersion lens. Images were taken every 2 µm through the embryo.