Dmi8 fluorescent microscope

After euthanizing mice, ear tissue was removed under aseptic conditions, fixed in 10% buffered formalin for 24 hours, embedded in paraffin and 5‐micron sections were then cut and processed for Hematoxylin and Eosin (H&E) staining. For immunohistology, after euthanizing mice, ear tissue was excised under aseptic conditions and frozen in optimal cutting temperature (OCT) media (Tissue‐Tek). Five micron frozen sections were cut using a Microm HM 550 Cryostat (Thermo Scientific), collected on coated slides, fixed in paraformaldehyde, washed with PBS, and blocked with appropriate sera in PBS. After incubating with appropriate antibodies, sections were washed and incubated with fluorescence dye‐conjugated second antibodies and 1 µg/mL of DAPI. Stained sections were washed and mounted under a coverslip using Fluoro‐gel with Tris Buffer (Electron Microscopy Sciences) and examined using a DMi8 fluorescent microscope (Leica). The measurement of ear thickness was carried out as described.20

TR146 cells were treated as described above. Cells were fixed with 4% paraformaldehyde followed by permeabilisation with 0.02% Triton-X for 20 min. After washing with PBS, the cells were blocked with 5% bovine serum albumin (Sigma-Aldrich, Missouri, USA) for 1 h at room temperature, followed by incubation with Nrf-2 antibody (Abcam) overnight at 4 °C. Subsequently, the wells were washed and incubated with goat anti-rabbit IgG-Alexa Flour 488 (Abcam) for 1 h and counterstained with 4′6-diamidino-2-phenylindole, DAPI (Abcam) and Phalloidin-iFlour 594 reagent (Abcam) for 5 min each. Stained wells were analysed using a Leica DMi8 fluorescent microscope.

RNA in situ hybridization was performed using the Multiplex Fluorescent V2 Assay kit (ACDBio) in combination with Opal Dyes (Akoya Biosciences) as per manufacturer’s instructions. The in situ probes are listed in the (Additional file 1: Table S3). Images of the optic nerve chiasm were acquired on an ICC50W fluorescent microscope with Leica Application Suite X software or with a Leica DMi8 fluorescent microscope using LAS X software.

Migration was measured by seeding 20,000 HASMCs into silicon inserts with a 500 μm gap in a 6-well culture plate for 24 h. Cells were then starved in serum-free media for 24 h to synchronize cells. After removing inserts, complete media supplemented with 10 μg/ml of mitomycin C (Sigma–Aldrich) were placed on HASMCs. Bright field images were captured at 0, 12, and 24 h using a Leica DMi8 Fluorescent Microscope equipped with a 4× objective. Percent gap closure was calculated by normalizing the size of the gap area between 0 and 12 or 24 h by the area at 0 h.
To measure proliferation, 2000 HASMCs were seeded in each well of a 96-well tissue culture plate. For time 0 recording, three wells from each condition were then washed with PBS and stained with CyQUANT Cell Proliferation Assay (Thermo Fisher Scientific) dye for 1 h at 37 °C, 5% CO₂, and 100% humidity after 1-h postseeding. A plate reader (Flexstation 3) was used to record fluorescence from these wells at 485 and 530 nm excitation and emission, respectively. The background fluorescence was subtracted from all readings. Plates were then returned to 37 °C, 5% CO₂, and 100% humidity for further readings at 24, 48, and 72 h. Readings were normalized to time 0 recording and represented as relative fluorescent unit/relative fluorescent unit₀.

At 0, 1, 2, 4 h time points after 12.5 μg/mL AMP treatment, E. coli samples were sampled and added to fresh LB medium supplemented with 0.5 μM of propidium iodide (PI) dye. After a 5-min incubation at 37 °C, cells are washed by PBS for three times, followed by fixation with 4% PFA for 15 min. We visualized and quantified the numbers of PI-positive cells and total cells, respectively. We captured images using Leica DMi8 fluorescent microscope.