Fluorochrome conjugated secondary antibody

Western blotting was performed using Run Blue precast native Page gels (Expedeon (https://www.expedeon.com/) Over, Cambridge, United Kingdom). Protein transfer was performed with the XCell II blot module (ThermoFisher Scientific). Polyvinylidene difluoride (PVDF) membranes were incubated with antibodies overnight/4°C and visualized using fluorochrome‐conjugated secondary antibodies (ThermoFisher Scientific) and digitally imaged.

Lungs and hearts were fixed with 4% paraformaldehyde for 24 h and embedded in paraffin. Sections (5 μm thick) were subjected to H&E staining. For immunofluorescence staining, antigen retrieval was performed by boiling the samples in citric acid buffer (pH 6.0) for 30 min. The sections were blocked in 5% goat serum for 60 min and incubated with primary antibodies (BM0002, Boster Biotech) at 4 °C overnight. This was followed by incubation with fluorochrome-conjugated secondary antibodies (Thermo Fisher Scientific) at room temperature for 2 h and DAPI (P36931, Thermo Fisher Scientific) counterstaining. WGA staining was performed at 37 °C in the dark for 2 h. Wall thickness in experimental PH models was assessed using vessels (<100 μm in diameter) and bronchial arteries were excluded. Wall thickness was calculated with the formula ((2×medial wall thickness/external diameter) ×100).^{59 (link)} The sections were inspected using a fluorescence microscope (Leica).

Paraffin-embedded kidney tissue sections were deparaffinized and subjected to antigen retrieval, which was performed under high pressure in citrate buffer (0.01 mol/L, pH 6.0) for 10 min. After blocking with 5% bovine serum albumin (BSA) for 1 h, the sections were incubated with primary antibodies overnight at 4°C (Nrf2, GeneTex, USA, #GTX103322; synaptopodin, Progen, Germany, #65194), followed by incubation with fluorochrome-conjugated secondary antibodies (Thermo Fisher Scientific) at 37°C for 2 h in the dark. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI, Antgene, China). All images were taken with a fluorescence microscope (Olympus, Japan).

Macrophages were seeded and cultured on glass coverslips in 24-well plates. After treatment, the cells were washed twice with PBS, fixed with 4% paraformaldehyde (PFA) for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and blocked in PBS containing 1% BSA for 1 h. The cells were then incubated overnight with primary antibodies (1:100 dilution) at 4 °C, followed by incubation with fluorochrome-conjugated secondary antibodies (1:500 dilution, ThermoFisher) for 1 h at room temperature. After wash, nuclei were counterstained with DAPI containing mounting medium and the cells were imaged using the Olympus IX73 Microscope System.

Formalin-fixed post-mortem brain tissues were obtained from brains donated to New York Brain Bank at Columbia University. All participants consented to brain donation at the time of death. The average age of death for all subjects in the study was 70 years old, with AD subjects meeting the criteria for pathologic AD by the NIA Reagan criteria (score 1–2). Cognitively unimpaired subjects without AD pathology were selected as control subjects (NIA Reagan score 3–4) (Suppl.Table.1.b).
Six μm sections of formalin-fixed paraffin-embedded tissue from the frontal cortex (n = 3) were used to stain for NeuN (Millipore), ALDH1L1 (eBioscience), CD45 (Novus Biological), along with anti-BIN1 antibodies provided by Biogen.
Immunohistochemistry was performed using citrate for antigen retrieval. The sections were blocked with blocking medium (8% of horse serum and 3% of BSA) and incubated overnight at 4 °C with primary antibodies. Sections were washed with PBS and incubated with fluorochrome conjugated secondary antibodies (Thermo Fisher) and coverslipped with anti-fading reagent with DAPI (P36931, Life technology). Photomicrographs are captured at 20x magnification using Zeiss Axio Observer Z1 fluorescence microscope and exported to Image J imaging software [23 (link)] (NIH, Maryland, USA). The images were quantified using CellProfiler and CellProfiler Analyst [24 (link)].

Mice were euthanized 1 day after MCAO. Brains were removed following perfusion with saline and 4% paraformaldehyde (Biosharp, Guangzhou, China) in phosphate buffered saline (PBS) and then soaked in 30% sucrose in PBS. The brains were embedded in OCT (Sakura Finetek, United States) solution after sinking to the bottom of the liquid, and cryosections were prepared. Then brains were cut on a freezing microtome into 25 μm-thick sections and subjected to immunofluorescence staining. After antigen retrieval treatment, sections were incubated with a blocking buffer containing 5% normal goat serum and 0.3% Triton X-100 (Thermo Fisher Scientific) at 37°C for 1 h. Then the sections were incubated with anti-Iba1 (ab178846, Abcam, United States) or GFAP (16825-1-AP, Proteintech, United States) at 4°C overnight, followed by Fluorochrome-conjugated secondary antibodies (Thermo Fisher Scientific) at room temperature for 2 h the next day. Finally, the sections were counterstained with DAPI (Thermo Fisher Scientific). Images were captured using a fluorescence microscope (Leica, Germany).