Prolong glass antifade mountant with nucblue stain

Zebrafish ectoderm cell aggregates were fixed in 4% paraformaldehyde in PBS for 30 min and per- meabilized in 0.1% Triton X-100 in PBS for 10 min. The non-specific binding sites were blocked by incubation in a culture medium containing 10% serum for 2 h. Primary antibody against phosphorylated (pS19) myosin light chain (rabbit, polyclonal, Anti-MYL12A phospho S19, Abcam ab2480) was applied in 1/100 dilution for 2 h at room temperature and then overnight at 4 °C. Anti-rabbit secondary antibody conjugated with AlexaFluor-555 (Southern Biotech, 4030-32) was used in 1/200 dilution for 4 h at room temperature. All incubations were followed by triple washing steps in PBS for 1 h. Finally, immunolabeled aggregates were mounted on microscopic slides (Thermo Scientific) using a mounting medium (Prolong Glass Antifade Mountant with NucBlue Stain, Invitrogen, P36981) containing NucBlue counterstain to visualize cell nuclei. Fluorescent labels were imaged using a Zeiss Axio Observer Z1 microscope with Zeiss EC Plan-Neofluar 40x/0.75 or Olympus A 100x/1.3 objectives and Zeiss AxioCam MRm CCD camera. Images were processed using NIH ImageJ software.

Monoculture and co-culture cells were incubated in 6-well dishes on glass coverslips. Then, 24 h following the radiation treatment, the samples were rinsed with PBS, and then fixed with 4% PFA for 5 min. After being fixed with PFA, the cells were rinsed with PBS, then washed with 2% BSA/0.1% Triton-X and incubated for 20 min. DNA DSBs damage was assessed using an optically labeled antibody against the repair protein, 53BP1. The 53BP1 primary antibody was diluted 1:200 in 0.5% BSA/0.1% Triton-X/PBS, whereas the secondary antibody was diluted 1:500 in 0.5% BSA/0.1% Triton-X/PBS. The samples were initially incubated with the primary antibody for 1 h, then rinsed with PBS. After that, the samples were washed with 0.5% BSA/0.175% Tween-20/PBS for 5 min and incubated with the secondary antibody for 30 min in the dark. The samples were then washed with PBS and mounted onto glass coverslips using ProLong™ Glass Antifade Mountant with NucBlue™ Stain (Invitrogen, Waltham, MA, USA). A 60× oil immersion lens was used to perform imaging of the 53BP1 foci through confocal microscopy (Zeiss LSM 980, Carl Zeiss AG, Jena, Germany). A minimum of 50 nuclei were assessed, and the number of foci per cell was measured.

Vero E6 cells were seeded on coverslips in 1× DMEM (5% FBS) and infected the following day with CORAVAX™ or control RABV (BNSP333) in 1X DMEM (2% FBS) at 34 °C for 48 h. At the end of the incubation, the cells were washed with 1X PBS, fixed in 2% PFA for 30 min, and blocked with PBS containing 5% FBS. The cells were then stained for 2 h at RT with mouse polyclonal antiserum against the S1 subunit of SARS-CoV-2 Spike protein and a human monoclonal antibody 4C12 against RABV glycoprotein (2 µg/ml). Following washing with PBS and incubation with Cy3-conjugated anti-rabbit IgG HRP (1:200 in PBS containing 5% FBS, Jackson ImmunoResearch, West Grove, PA, Cat# 711-035-152) and Cy2 conjugated anti-human IgG HRP (1:250 in PBS containing 5% FBS, Jackson ImmunoResearch, Cat# 109-225-088) secondary antibodies. The cells were mounted in ProLong™ Glass Antifade Mountant with NucBlue™ Stain (Invitrogen, Cat# P36983). Images were taken using Nikon A1R+ confocal microscope. Composite images were prepared using Fuji.

Cells were plated on acid‐washed coverslips coated with or without 10 µg/mL fibronectin at 37 °C for 1 h. Cells were then fixed with 4% paraformaldehyde at room temperature for 15 min, permeabilized in 0.5% Triton X‐100 in DPBS for 8 min, and blocked with 5% bovine serum albumin (BSA) for 1 h. To stain target proteins, the primary antibodies were diluted 1:200 in DPBS and incubated for 1 h at room temperature. After washing with DPBS three times, the coverslips were incubated with Alexa Fluor 488, 555, or 647‐conjugated secondary antibody for 1 h at room temperature. After another wash with PBS three times, the coverslips were mounted with ProLong Glass Antifade Mountant with NucBlue Stain (P36981, Invitrogen). After mounting medium was solidified, images were captured by Andor Dragonfly confocal imaging system and FACILITY STED microscopy (Abberior).

Identity of migrated HFSCs was assessed by immunostaining against nestin as a neural crest stem cell marker and SOX10 as a neural crest cells marker [6 (link)] using mouse anti-nestin (1:50; Abcam, #ab6142) and rabbit anti-SOX10 (1:100; proteintech, 10422-1-AP) primary antibodies. Briefly, cells at passage 1 were seeded in a 4-well chambered cell culture slide and fixed with 4% paraformaldehyde. Following several washing steps, cells were blocked with 10% normal goat serum, 1% FBS and 0.1% Triton X-100 prepared in phosphate-buffered saline (PBS). Then, the primary antibodies were applied overnight at 4 °C. After washing cells and re-blocked with 3% bovine serum albumin for 10 min, cells were exposed to goat anti-mouse IgG AlexaFluor488 (1:1000, ThermoFisher, #A-11001) or goat anti-rabbit IgG AlexaFluor488 (1:1000, Abcam, #ab150085) secondary antibodies at room temperature for two hours. To counterstain the nuclei, the ProLong™ Glass Antifade Mountant with NucBlue™ Stain (Invitrogen, # P36985) was used to cover the chambers. Finally, immunofluorescent images were obtained using a Leica DM5000B epifluorescence microscope.

CuO-POTEF-Flag HGrC1 cells were fixed with MeOH, permeabilized with 0.5% Triton X-100 in PBS, blocked in 1% bovine serum albumin (BSA)/PBS, and stained with the following antibodies: mouse: anti-TCP-1α (sc-374088), rabbit: anti-LC3 (PM036, MBL), and anti–DDDDK-tag mAb-Alexa Fluor 488 (M185-A48). Alexa Fluor-conjugated species-specific anti-IgG (Thermo Fisher Scientific) in 1% BSA/PBS was used as the secondary antibody. Images were obtained using BZ9000 microscopy (Carl Zeiss Microscopy Co., Ltd.). Secondary antibodies were conjugated with Alexa Fluor 594 for TCP-1α and for LC3. We set the colors of Alexa Fluor 488 to green and Alexa Fluor 594 to red. Nuclei were stained blue using ProLongGlass Antifade Mountant with NucBlue Stain (Invitrogen).