Alexa fluor 546 donkey anti goat igg

Immunostaining was conducted on isolated islets, fixed in 4% paraformaldehyde before being permeabilised using 0.1% Triton X-100 (Sigma). After blocking with 5% goat serum, islets were incubated overnight (4°C) with primary antibodies before incubation with secondary antibodies. Fluorescent staining was visualised using a laser-scanning confocal microscopy (BioRad) controlled by LaserSharp2000 (BioRad).
Antibodies used in this study were: rabbit FITC 495-conjugated anti-GFP (1:250; DS-PB-00926; InsightBio, Wembley, United Kingdom), mouse anti-glucagon (1:1000; G2654; Sigma), guinea-pig anti-insulin (1:400; A0564; Dako, Santa Clara, California, United States), goat anti-somatostatin (1:100; sc-7819; Santa Cruz Biotechnology, Dallas, Texas, United States); Alexa Fluor 633 goat anti-mouse IgG (1:500; A-21052; ThermoFisher); Alexa Fluor 594 goat anti-guinea pig IgG (1:500; A-11076; ThermoFisher); and Alexa Fluor 546 donkey anti-goat IgG (1:100; A-11056; ThermoFisher).

Ileum tissues were fixed with 4% PFA and dehydrated with 15%, and then 30% sucrose in PBS. Dehydrated tissues were formed into a Swiss roll, frozen, and sliced. For staining, collected organoids were permeabilized in PBS containing 0.1% Tween 20 and blocked with 0.5% BSA in PBS for 1 h. For Muc2 staining, ileum tissues containing feces were fixed in Carnoy’s solution and embedded in paraffin. The primary antibodies used were rabbit anti-Muc2 (Abcam), rat anti-Ki67 (Biolegend), rabbit anti-lysozyme (Abcam), goat anti-Wnt3 (Abcam), mouse anti-β-catenin (BD Bioscience), and mouse anti-phospho-ERK1/2 (Thermo Fisher Scientific). Secondary antibodies were Alexa Fluor goat 594 anti-rat IgG (Biolegend), Alexa Fluor 488 goat anti-mouse IgG (Abcam), Alexa Fluor 546 donkey anti-goat IgG (Thermo Fisher Scientific), and Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific).

NHEKs and NHEMs were seeded at 5 × 10⁴ cells/dish (35 mm dish), incubated for 72 hrs, and fixed with 4% paraformaldehyde for 10 min. After the cells were permeabilized with 0.2% triton for 5 min, they were blocked using a mixture containing 1% BSA and 20% heat-inactivated serum for 30 min. The cells were then incubated overnight at 4 °C with Human Osteoactivin/GPNMB Antibody (1:200, antigen affinity-purified polyclonal goat IgG, catalog number: AF2550; R&D Systems, MN, USA) as the primary antibody. Next, the cells were incubated with Alexa Fluor 546 Donkey Anti-Goat IgG (1:500, Thermo Fisher Scientific, MA, USA) as the secondary antibody at room temperature for 1 hr. After incubating the cells with Hoechst for 5 min, images were taken using a confocal microscope (Olympus, Tokyo, Japan).

Mouse lung cells were fixed, blocked and permeabilized with BD Cytofix/Cytoperm Kit, according to the manufacturer’s instructions. Cells were then incubated with goat anti‐mouse pro‐surfactant protein C) (proSP‐C; Santa Cruz Biotechnology, Dallas, TX, USA) or rat anti‐(mouse CD31) (BD Pharmingen) IgG at 4 °C overnight and then incubated for 1 h with Alexa Fluor 546 donkey anti‐(goat IgG) or Alexa Fluor 546 goat anti‐(rat IgG) (Molecular Probes, Carlsbad, CA, USA), respectively.

Serial 40-μm-thick sections were rinsed twice with 0.1 M phosphate-buffered saline (PBS) and washed in 0.1 M Tris buffer (pH, 7.6) containing 0.3% Triton X-100. Sections were incubated for 72 h at 4 °C in primary antibody solution (goat anti-c-Fos, Santa Cruz Biotechnology, TX, USA; 1:500 or rabbit anti-OXT, Sigma-Aldrich, MO, USA; 1:5000)^{65 (link)}. After washing twice in 0.3% Triton X-100 in PBS, floating sections were incubated for 24 h at 4 °C with a secondary antibody (Alexa Fluor 546 donkey anti-goat IgG or Alexa Fluor 488 donkey anti-rabbit IgG; Molecular Probes, OR, USA; 1:2,000 in PBS containing 0.3% Triton X-100)^{65 (link)}. Sections were washed twice in PBS and then mounted on the slides and coverslipped using vectashield (Vector Laboratories Co. Ltd., CA, USA)^{66 (link)}. Images of Fos⁺, OXT⁺, and Fos⁺/OXT⁺ double-labelled cells were counted manually by two researchers who were blinded to avoid bias. The number and percentage of Fos⁺, OXT⁺, and Fos⁺/OXT⁺ cells in the SON and PVN were estimated.

For whole-mount DAPI staining to visualize palate tissues (Sandell et al., 2012 (link)), E15.5 and E17.5 embryos were fixed in 4% PFA and incubated overnight in 1:1000 DAPI dilution (Dojindo).
Analyses of apoptotic cells were performed using an in situ cell death detection kit (www.Roche.com) following the manufacturer's instructions. For analyses of proliferation, samples were incubated with a mouse anti-pHH3 antibody (1:200, Millipore) at 4°C overnight followed by secondary Alexa-Fluor-488 donkey anti-mouse IgG (1:200, Invitrogen) for 6 h at room temperature for sections and overnight at 4°C for whole embryos. To label NCCs, sections were counterstained with a goat anti-Sox10 antibody (5 µg/ml, R&D Systems) at 4°C overnight, followed by secondary antibody (Alexa-Fluor-546 donkey anti-goat IgG, 1:200, Molecular Probes). Cells in at least five adjacent sections were counted in each assay. Statistical significance was assessed using a two-tailed Student's t-test.