C1 laser scanning confocal microscope

Alkaline phosphatase staining (Supplementary Fig. S1a) was performed using the Leukocyte Alkaline Phosphatase kit (Sigma).
The cultures of iPSCs and MNs were immunostained following standard procedures39 (link). Briefly, cells were fixed in 4% paraformaldehyde for 15 min at 4 °C, washed with PBS, and incubated in a blocking buffer (10% donkey serum and 0.2% Triton X-100 in PBS) for 60 min at room temperature before being incubated in primary antibodies (Supplementary Table S2) overnight at 4 °C. Appropriate fluorescently conjugated secondary antibodies were used to reveal the binding of primary antibodies (1:1000, Jackson, West Grove, PA) and nuclei were stained with DAPI. Images were collected with a Nikon TE600 fluorescence microscope (Nikon Instruments, Melville, NY) or a Nikon C1 laser-scanning confocal microscope (Nikon, Tokyo, Japan).
To quantify the population of OLIG2⁺, MNX⁺, and ChAT⁺ cells among total cells (DAPI labeled) or neurons (TuJ1⁺), images were imported into ImageJ (NIH) for analysis. Cell counting was performed by a person blind to the experiment and replicated in different cell lines in three independent experiments.

C57BL/6J mice were deeply anaesthetised with sevoflurane, and testicles were collected from adult (P60 ± 10 days) Kcnj16^(−/−) and Kcnj16^(+/+) male mice. The testis and epididymis were dissected out and immediately fixed by immersion in 4% paraformaldehyde in phosphate buffer (PB, pH 7.4) overnight. The tissues were then stored in 30% sucrose in PB overnight. Cryostat sections (15 μm) were collected on gelled slides, rinsed in phosphate buffer saline (PBS, pH 7.4), and then incubated overnight in rabbit anti-Kir4.1, anti-Kir4.2, and anti-Kir5.1 antibodies (Alomone labs, Jerusalem, Israel, cat#: APC-035, APC-058, APC-123) diluted 1:1000 in PBS containing 0.3% Triton. After rinsing with PBS, the sections were incubated with anti-rabbit conjugated to Alexa 488 diluted 1:200 in PBS (Jackson Immuno Research, West Grove, PA, USA) for 1 h. Sections were mounted in antifade immuno-mount (Thermo Fisher Scientific, Runcom, Cheshire, UK) and examined with a Nikon fluorescent microscope equipped with appropriate filters and a Nikon C1 laser scanning confocal microscope (Nikon, Tokyo, Japan). Representative digital images were captured and the resulting files were used to generate figures in Adobe Photoshop software CS6 (San Jose, CA, USA) where photomicrographs were adjusted for contrast and brightness.

Wandering third instar larvae from sparsely populated bottles were collected and dissected in Ca2+-free saline. A detergent-free protocol was used to visualize labeling at the NMJ for anti-Sdc (1:250) [20 (link)], but all other antibodies including anti-HRP [1:2000] (Jackson Immunoresearch), anti-FasII [1:20] (Developmental Studies Hybridoma Bank), and anti-myc [1:3000] (Developmental Studies Hybridoma Bank) were used in the presence of 0.1% Triton as previously described [21 (link)]. Alexa488 goat anti-mouse, Alexa488 goat anti-rabbit, Alexa568 goat anti-rabbit and Alexa568 goat anti-mouse secondary antibodies (Invitrogen) were used at 1:500. Imaging was done on a Nikon C1 laser scanning confocal microscope. Statistical analysis of boutons per NMJ was conducted in Excel by Student’s t-test on larval pelts that were scored blind to genotype.

Immunofluorescence analysis was used to detect Treg cells in MC38 tumor tissues extracted from mice. The details of the procedure have been described (46 (link)). FFPE tissues were sectioned and transferred to gelatin-coated slides. Tissue sections were dewaxed, rehydrated, blocked with 3% BSA and incubated overnight with the primary antibodies: rabbit anti-CD4 (1/1000, ab183685, Abcam) and rat anti-Foxp3 (1/50, 14-5773-82, eBioscience, San Diego, CA, USA). Following a washing step, sections were incubated with anti-rat Alexa Flour-488-labelled (1/200, 712-545-153, Jackson ImmunoResearch, West Grove, PA, USA) and anti-rabbit TRITC-labelled (1/100, 111-025-003, Jackson ImmunoResearch) secondary antibodies for 1 hour in the dark. TO-PRO™-3 iodide (642/661) (T3605, Thermo Fisher Scientific) was used as a counterstain for viable cell nuclei. Last, sections were washed, mounted with fluorescence medium (Dako) and visualized using Nikon C1 laser scanning confocal microscope.