Fv1000 confocal microscope

The embryos were washed three times with phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA, Sigma-Aldrich) and fixed in 4% paraformaldehyde for 30 min. Fixed embryos were washed three times in PBS containing 0.5% BSA, used immediately, or stored at 4°C in embryo storage buffer (PBS + 0.9% sodium azide) for up to 1 week. Fixed embryos were permeabilized with 0.01% Triton X-100 for 30 min and washed three times with PBS containing 0.5% BSA before being blocked in a 5% BSA/PBS solution for 1 h. Embryos were incubated with a primary antibody against BMP2 at a 1:500 dilution in 5% BSA/PBS overnight at 4°C. Finally, these embryos were washed three times for 20 min in 0.5% BSA/PBS containing 0.05% Tween 20 (0.5% BSA/PBST) and incubated with a fluorescein isothiocyanate-conjugated secondary antibody (1:200, Invitrogen, Carlsbad, CA, United States) for 1 h. The nuclei were stained with 1 mg/mL 40′, 6′-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 10 min. Embryos at the 8-cell stage were chosen as negative controls. Images were taken on an Olympus FV1000 confocal microscope and processed using Adobe Photoshop. The same experiment was independently repeated three times, each time in triplicate, and 20–30 embryos of different developmental stages were examined each time.

On 21 d after STZ injection, rats were anesthetized with pentobarbital sodium and transcardially perfused with normal saline succeeded by 4% paraformaldehyde in 0.1 M phosphate buffer saline (PBS). DRGs from L3-5 were isolated, postfixed overnight in 4% paraformaldehyde in 0.1 M PBS, and cryoprotected in 30% sucrose in 0.1 M PB at 4°C till the tissue sinkage to the bottom of the container. Transverse DRG sections (14 μm) were sliced on a cryostat and mounted serially on slides. To visualize HCN2 channels, immunofluorescence labeling methods were used. Briefly, the sections were rinsed twice with 0.1 M PBS and then incubated with a solution containing 0.3% Triton X-100 and 1% donkey serum albumin for 3 h at r/t. The sections were thereafter incubated with anti-HCN2 channel antibodies (1 : 200, rabbit anti-HCN2, Alomone Labs) in PBST for 24 h at 4°C. After three washes with PBST, the sections were further incubated with secondary antibody Alexa Fluor 488 (1 : 200, donkey antirabbit IgG, Invitrogen, USA) for 2 h at r/t. The sections were then mounted with antifade medium and stored at 4°C. All images were captured with Olympus FV1000 confocal microscope and processed with Adobe Photoshop software. The negative controls were performed using the same procedure without addition of primary antibody.

To evaluate the monolayer integrity of MTECs ALI14, Transwells were put on ice and their upper chamber was washed twice in cold Ca/Mg-PBS and incubated with 0.5 mg/ml sulfo-NHS-LC-Biotin (Thermo Scientific) twice for 20 min at 4°C. Then, MTECs were fixed in 4% PFA for 30 min, washed three times in PBS, and stained with Alexa-Fluor 555 streptavidin (Invitrogen, #S21381, 1:500), DAPI at 0.5 μg/ml, and Alexa-Fluor 488 phalloidin (Invitrogen, #A12379, 1:500). Samples were washed three times in PBS and mounted in Vectashield. Finally, images were obtained using an Olympus FV 1000 confocal microscope and were processed using ImageJ and Adobe Photoshop CC 2019.