Hb050 inverted microscope system

Immunofluorescence staining was performed according to our previously published protocols [41 (link), 42 (link)]. Briefly, the brain sections (6 μm thick) were incubated with primary antibodies against ALX/FPR2 (1:200, cat. no. ab203129, Abcam), GFAP (1:200, cat. no. MAB3402X, EMD Millipore, USA), Iba-1 (1:200, RRID: AB_2224402), and NeuN (1:200, cat. no. ABN78A4, EMD Millipore, USA) overnight at 4 °C. Then, the brain sections were incubated with the corresponding secondary antibodies conjugated with Alexa Fluor 488 and/or Alexa Fluor 594 (Jackson ImmunoResearch Incorporation, West Grove, PA, USA) for 1 h at room temperature, after which a ZEISS HB050 inverted microscope system was used for fluorescence detection.
For in vitro cell staining, primary antibodies against Iba-1 (1:200, RRID: AB_2224402) and p65 (1:200, 1:1000, cat. no. D14E12, CST) were used for microglia staining, while a primary antibody against MAP2 (1:500, cat. no. ab183830, Abcam) was used to stain neurons. The protocols and the secondary antibodies used for in vitro cell staining were the same as those used to stain brain sections.

Immunofluorescence staining was performed according to our previously published Protocols (Sun et al., 2014 (link); Ye et al., 2018 (link)). Briefly, brain sections (6 μm) were first incubated with primary antibodies against p65 (1:200, 1:1000, cat. No. D14E12, CST), iba-1 (1:200, RRID: AB_2224402) overnight at 4 °C. The next day, brain sections were incubated with corresponding secondary antibodies Alexa Fluor 488 and/or Alexa Fluor 594 (Jackson ImmunoResearch Incorporation, West Grove, PA, United States). Fluorescence was examined under a ZEISS HB050 inverted microscope system. The fluorescently stained cells were analyzed by Image J software.

Fresh proximal aorta attached to heart were immediately embedded in Tissue-Tek OCT compound (Sakura Finetech Japan, Tokyo, Japan) and snap-frozen. Unfixed frozen samples were cut into 6-μm-thick sections and placed on glass slides. Dihydroethidium (DHE; 10 μM, Sigma-Aldrich) was applied to each tissue section, and the slides were subsequently incubated at 37°C in the dark for 30 min. Images were immediately obtained using a ZEISS HB050 inverted microscope system (Zeiss, Jena, Germany). Assessment of relative ATP contents in aorta was performed using the ATP bioluminescence assay kit (Beyotime, Haimen, China) following the manufacturer’s instruction. Aorta superoxide dismutase (SOD) activity was analyzed with a SOD assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Intracellular ROS contents were determined using the fluorescence probe dihydroethidium (DHE) according to the instructions of the manufacturer (Sigma-Aldrich, St. Louis, MO, United States). Briefly, the frozen sections were incubated in DHE and then treated with PBS for 30 min at 37°C in the dark. The sections were washed three times for 5 min in PBS (pH 7.4). Immunofluorescence imaging was performed using a ZEISS HB050 inverted microscope system. DHE-fluorescence intensity (representing the intracellular ROS levels) was measured using the Image-Pro Plus 6.0 software.
Production of mitochondrial ROS was detected using a MitoSOX^TM Red mitochondrial superoxide indicator (Thermo Fisher Scientific, Waltham, MA, United States). Fresh temporal cortex tissues weighing 10 mg were dispersed to single-cell suspension using a pipettor. The cells were washed twice with PBS and diluted in 5 mM MitoSOX^TM reagent stock solution in HBSS/Ca/Mg buffer to make a 5 μm MitoSOX reagent working solution. A working solution of 1.0 ml 5 μm MitoSOX reagent was applied to incubate the cells for 10 min at 37°C in the dark. Fluorescence was detected using a microplate reader at an emission wavelength of 510 nm and an excitation wavelength of 580 nm.

The brain sections with antigen retrieval or cultured cells were treated with QuickBlock™ Blocking Buffer (cat# P0260, Beyotime, China) for 30 min at room temperature and then incubated with primary antibodies against Nrf2 (1:100, cat# ab137550, Abcam), MAP2 (1:200, cat# sc-32791, Santa Cruz), NeuN (1:200, cat# MAB377, EMD Millipore), and Iba-1 (1:100, cat# ab178846, Abcam) overnight at 4 °C. The cells or slides were incubated with corresponding secondary antibodies Alexa Fluor 594 and/or Alexa Fluor 488 (1:200, Jackson ImmunoResearch Incorporation, West Grove, PA, USA). Then, the cells or slides were washed in phosphate buffered saline-Tween twice for 20 min and counterstained with 4,6-diamidino-2-phenylindole (DAPI) (1:2000, cat# ab104139, Abcam) for 5 min. Fluorescence was captured on a Zeiss HB050 inverted microscope system. The ImageJ software was used to measure the inflorescence intensity.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining
was conducted by using a TUNEL detection kit according to the manufacturer's instructions (Roche Inc., Indianapolis, USA). Briefly, each section was incubated with primary antibody against NeuN (1:100, Millipore, MA, USA) at 4 °C overnight. After sections were washed three times with PBS for 45 min, they were incubated with TUNEL reaction mixture for 1 h at 37 °C in the dark. The slides were washed with PBS again three times for 45 min prior to be counterstained by DAPI for 2 min. After three washes again, the slides were covered by microscopic glass with anti-fade mounting medium for further study. Fluorescence microscopy imaging was performed using ZEISS HB050 inverted microscope system. The positive cells were identified, counted and analysed by two investigators blinded to the grouping. The extent of brain damage was evaluated by the apoptotic index, defined as the average percentage of TUNEL-positive cells in each section counted in 10 cortical microscopic fields (at × 400 magnification). A total of four sections from each animal were used for quantification. The final average percentage of TUNEL-positive cells of the four sections was regarded as the data for each sample.

Immunofluorescence staining was carried out as described previously (Sun et al., 2014 (link)). Frozen tissue sections (6 μm thickness) were incubated in blocking buffer for 2 h at room temperature. After they were washed three times with PBS, sections were incubated with primary antibodies and corresponding secondary antibodies. After washing with PBS for three times, the slides were stained with 4-diamidino-2-phenylindole (DAPI) for 2 min. Images were obtained using a ZEISS HB050 inverted microscope system and the fluorescently stained cells were analyzed using Image J program.