Ti2 confocal microscope

Protein interactions between GHSR/DRD1 in mouse brain slices were detected using Duolink PLA detection kits (Sigma-Aldrich, DUO92008) following manufacturer’s instructions. The following primary antibodies were used: GHS-R1a (Santa Cruz Biotechnology, sc-10359, 1:100) and anti-DRD1 (Abcam, ab81296, 1:200). The specificity of antibodies to GHSR and DRD1 was validated as previously described (17 (link)). The following Duolink in Situ PLA Probes were used: anti–rabbit PLUS (Sigma-Aldrich, DUO92002) and anti–goat MINUS (Sigma-Aldrich, DUO92006). Images were collected on a Nikon Ti2 confocal microscope. The mean intensity of PLA⁺ signals was analyzed using Nikon-Elements Advanced Research software.

Brain slices were washed with PBS at room temperature for 20 min, followed by antigen retrieval at 85 °C for 30 min using 10 mM sodium citrate buffer. Slices were permeabilized with 0.4% Triton X-100 in PBS at RT for 20 min. The slices were then blocked with 10% donkey serum and 0.1% Triton X-100 in PBS at RT for 3 h, followed by incubation with primary antibodies, 5% donkey serum, and 0.1% Triton X-100 in PBS at 4 ◦C overnight. After washing three times with 0.1% Triton X-100 in PBS at RT for 15 min, secondary antibodies, 5% donkey serum, and 0.1% Triton X-100 in PBS were added for 2 h at 4 °C. The slices were counterstained with DAPI and mounted with a mounting medium (Vectashield, Vector Laboratories Inc., Burlingame, CA, USA). The following antibodies were used: rabbit anti-TREK1 (Alomone Labs, Jerusalem, Israel, 1:200; RRID: APC-047, 1:200), mouse anti-β-COP (Santa Cruz Biotechnology, D-10, 1:200), rat anti-GFAP (Thermo Fisher, Waltham, MA, USA; 1:500, RRID:13-0300, 1:500), and Alexa Fluor 488-, 594-, and 647-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA; 1:300). All images were acquired using a Nikon Ti2 confocal microscope (Nikon Instruments, Inc., Melville, NY, USA).

F-actin staining was conducted to assess cell morphology using Actin-tracker Red-555 (diluted 1:100) and DAPI (both from Beyotime). Fluorescence images were captured using a Nikon Ti2 confocal microscope.

For each transfection, 50 000 cells were seeded onto each glass slide. The cells were transfected with 1ug of each indicated plasmid and fixed and stained 24 h post transfection. Primary antibodies were incubated for overnight at 4°C (cell signaling anti-FLAG 9A3, cell signaling anti-T7 D9E1X and secondary antibodies (Alexa Fluor 488 and Alexa Fluor 488) were incubated for 1 h at room temperature. Slides were mounted with DAPI (4′,6′-diamidino-2-phenylindole), and confocal analysis was performed using Nikon Ti2 confocal microscope.

FISH was performed using an RNAscope Fluorescent Multiplex Kit (Advanced Cell Diagnostics, RRID; SCR_012481, CA, USA) and a Kcnk1-C1 probe (Advanced Cell Diagnostics, Cat#; ACD 535421, CA, USA) according to the manufacturer’s instructions. Subsequently, 15-μm fresh frozen brain slices were fixed with 4% PFA for 15 min and then washed in 50% (×1), 70% (×1), and 100% (×2) ethanol for 5 min each. After air drying for 5 min, the slices were digested with protease solution at RT for 30 min, followed by washing with PBS three times. The pre-warmed probe was applied to the slices, which were incubated in a humidified oven at 40 °C for 2 h. Slices were washed twice with the wash buffer and then amplified with AMP 1 to AMP 4, followed by counterstaining with DAPI. Slices were mounted with ProLong Gold Antifade (Thermo Fisher Scientific, Cat#; P36930, MA, USA). Images were acquired with a Ti2 confocal microscope (Nikon, RRID; SCR_021068, Tokyo, Japan). Z-stack images were acquired using a Nikon Eclipse Ti2 confocal microscope (Nikon, RRID; SCR_021068, Tokyo, Japan) using a Plan Apo Lambda 40x/0.95 objective (Nikon, Material#: MRD00405, Tokyo, Japan) with a 0.6232 × 0.6232 × 2 μm³ voxel size. Kcnk1 mRNA spots were manually counted from maximum z-projection images using Fiji software (Max Plank Institute of Molecular Cell Biology and Genetics, RRID: SCR_002285, Dresden, Germany).

Briefly, a 10% GelMA solution (in PBS) containing 0.3% lithium phenyl-2,4,6-trimethylbenzoyl phosphate (LAP) as photo-initiators was mixed with MSCs-laden GMs in a volume ratio of 1:2 at 37°C. The bead-suspended blend was transferred to a 1 ml syringe loaded on a LivPrint N bioprinter (Medprint) and used to fabricate a grid human-shaped model with a layer height of 0.4 mm. Extrusion speed was set to 0.09 mm/s while nozzle movement speed was set to 18 mm/s using a 20 G nozzle. The printing process was carried out at room temperature with ultraviolet light at 405 nm used for crosslinking. The scaffold was incubated in MSCM with media changes every 2 or 3 days. To assess the impact of the printing process on cell viability, live/dead staining was performed using calcein-AM and PI. Fluorescence images were captured using a Nikon Ti2 confocal microscope (capture depth 100 μm).