Laser confocal fluorescence microscope

We verified the purity of primary murine retinal microglia cultures and the localization of GPER in the microglia using immunofluorescence. Briefly, the retinal microglial cells were fixed with 4% paraformaldehyde, and then permeabilized with 0.5% Triton X-100 at room temperature for 15 min. Then, the cells were incubated with 6% normal goat serum (Hyclone, Logan, Utah, USA) at room temperature for 30 min for blocking. The slides were then incubated overnight with either mouse anti-CD11c antibody (1:200, Abcam, Cambridge, UK), a microglia specific marker located on their cell membrane [50 (link)], rabbit anti-GPER antibody (1:200, Abcam, Cambridge, UK), or PBS (negative control) at 4°C. The cells were then incubated with the Alexa Fluor 488-tagged secondary antibody (1:800, Abcam, Cambridge, UK) for 30 min at 37°C in the dark. Then, after staining the nuclei with DAPI (Beyotime, Shanghai, China), the slides were washed thrice with PBS to remove the excess DAPI. The slides were sealed with anti-fluorescence quencher (Beyotime, Shanghai, China) and imaged using a laser confocal fluorescence microscope (Leica, Solms, Germany). The average fluorescent intensity was measured using the Image J software (version 1.46, National Institutes of Health, Bethesda, MA, USA).

Paraffin sections of substantia nigra were dewaxed in water and incubated with 3% BSA and 0.3% Triton for 2 h at room temperature to block the antigen. After washing with PBS, the sections were incubated with rabbit anti-mouse Nrf2 antibody (1:400, Abcam, UK) at 4 °C for 24 h and Alexa Fluor488 treatment-labeled goat anti-rabbit IgG (1:400, Abcam, UK) at room temperature for 2 h in the dark. After washing with PBST, the sections were imaged with a laser confocal fluorescence microscope (Leica, Germany). To quantify the immunofluorescence intensity, the integrated optical density (IOD) was calculated using Image J software, and the mean IOD (MOD) was calculated from IOD/Area.

Neural stem cells treated with OGD were seeded in a 6-well plate at 1 × 10⁵ cells per well. After culturing overnight, the original medium was replaced with a fresh medium containing EdU (100 μM, Camilo, Nanjing, China) for 2 hours, then the cells were fixed with acetone for 20 minutes. The Apollo reaction buffer with FITC fluorescein was incubated for 1 hour and then photographed with a laser confocal fluorescence microscope (Leica, Wetzlar, Germany).

The changes of cells nuclei under nano Cu treatment were detected using Hoechst 33342 staining. Cells were exposed to various concentrations of nano Cu for 3 h. Then, after incubating with Hoechst 33342 dye solution for 20 min in the dark, the sample was observed under a laser confocal fluorescence microscope (Leica, Germany). All experiments were performed in triplicate. The percentage of apoptotic cells was calculated as followed: apoptotic cells (%) = (number of apoptotic nuclei) / (number of total cell nuclei) × 100.

Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay was performed in the paraffin sections of the lung tissues by using the Fluorometric TUNEL System from KeyGen Biotech in accordance with the manufacturer’s instructions. Sections were counterstained with Hoechst 33258 dye for nuclear staining and photographed using a confocal laser fluorescence microscope (Leica). TUNEL-positive cells were counted in six visual fields that were randomly selected from each slide by two blinded observers.

The tissue sections and cells cultured on coverslips were fixed in 4% para-formaldehyde, rinsed with PBS, incubated in 0.5% Triton X-100 for 20 min. and blocked nonspecific antigens with 10% serum. Samples were then incubated overnight at 4°C with primary antibodies including anti-α-smooth muscle actin (α-SMA), anti-Bcl-2-associated X protein (Bax), anti-Drp1, anti-p53-up-regulated modulator of apoptosis (PUMA), anti-surfactant-associated protein (SP-C) and anti-Snail (Santa Cruz Biotechnology, Watsonville, CA, USA). Subsequently, treated samples were rinsed with PBS and then incubated with secondary antibodies labelled with FITC (fluorescein isothiocyanate) and Cy3 for 60 min. at 37°C, followed by incubation with Hoechst 33258 dye. Serums of the same species as the primary antibodies were used as the negative control. Cover slips or tissue sections were mounted on neutral glycerin and photographed under a confocal laser fluorescence microscope (Leica) with LAS AF software. The mean fluorescence intensity was expressed in fluorescence units, and dual-positive cells were counted in six visual fields that were randomly selected from each slide by two blinded observers.