Anti fluorescence quencher

First, Dihydroethidium (Beyotime, China) needs to be dissolved in DMSO (Biosharp, China) and prepared into an appropriate stock solution of 5 mg/mL. Then, for probe labeling, slice sections are taken out and routinely dewaxed in water. Next, immunohistochemical pen strokes are performed, and tissue autofluorescence quenchers are added. Following this, 100 μL of staining solution (DHE‐1:1000 dilution) is added, and the sample is incubated in a light‐avoiding incubator at 37°C for 30 min. Afterwards, the staining solution needs to be removed, and the sample is washed three times with PBS and stained with DAPI (Beyotime, China) for 5 min. Then, the slice is washed with PBS 2–3 times, and an anti‐fluorescence quencher (Beyotime, China) is added to seal the sample. Finally, the sample is examined using a fluorescent microscope.

The frozen brain sections were rehydrated and treated with L.A.B. solution (Polyscience, Inc., Niles, IL, USA) for 20 min. They were then blocked with goat serum for 30 min, and incubated overnight at 4 °C with both the mouse-anti-Aβ (1:400) and rabbit-anti-NeuN (1:400, Cell Signaling Technology, Danvers, MA, USA) antibodies. Subsequently, sections were incubated in DyLight 594-labeled goat anti-mouse IgG and 488-labeled goat anti-rabbit IgG (1:400, Cell Signaling Technology, Danvers, MA, America) at room temperature for 1 h. Finally, the sections were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime Institute of Biotechnology, Beijing, China) and sealed with anti-fluorescence quencher (Beyotime Institute of Biotechnology, Beijing, China). The sections were observed and photographed using a confocal fluorescence microscope (Leica, SP8., Wetzlar, Germany).

Cells (3 × 10⁵ cells/well) from different treatment groups were inoculated in six-well plates, with non-adhered cells subsequently removed. Adhered cells were washed twice with PBS, fixed with 1 ml of 4% paraformaldehyde at room temperature for 15 min, washed three times with PBS, incubated with goat serum for 1 h and then placed in a wet box overnight with the primary antibody anti-ADAM10 (1:100, GTX104940, GeneTex). After washing with PBS, 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Beijing, China) was added dropwise and incubated for 5 min at room temperature in the dark, with excess DAPI washed away and the film sealed with a blocking solution containing an anti-fluorescence quencher (Beyotime) for observation under a fluorescence microscope (BX41, Olympus).

The indicated cells were fixed, permeabilized, blocked and incubated overnight with primary anti-Ki67 antibody (1:200, Abcam, UK) at 4 °C. Subsequently, the cells were incubated with secondary antibodies (1:100, ZSGB, China) for 1 h and stained with 1× Hoechst 33324 for 5 min at room temperature. The cell slides were mounted with anti-fluorescence quencher (P0126, Beyotime, China) and observed under a fluorescence microscope. For paraffin sections, they were deparaffinized, blocked and then incubated with primary anti-CD31 (1:200, R&D) and anti-von Willebrand factor (1:200, vWF, Abcam) antibodies at 4 °C overnight. Then, the sections were incubated with secondary antibodies (1:100, ZSGB, China) and stained with 1× Hoechst 33324 at room temperature. Subsequently, the sections were observed under a 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).