Bx512dp70

The lung tissue sections were deparaffinized by xylene and graded ethanol. Endogenous peroxidase was blocked by incubating in 3% H₂O₂-methanol at room temperature for 30 min. After washing in phosphate buffered saline (PBS), slides were incubated with 5% normal goat serum in PBS at 37°C for 30 min to prevent non-specific staining. The slides were then incubated with antibody directed to MPO (1: 100) diluted in PBS containing 1% bovine serum albumin overnight at 4°C. Specific binding was detected by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (ZSGB-BIO, Beijing, China) and revealed with the 3,3′-diaminobenzidine (DAB) substrate Kit. The images were captured by a digital camera connected to a microscope (BX512DP70, Olympus, Tokyo, Japan).

Following dewaxing in xylene and rehydration in ethanol, antigen retrieval was performed in 0.01 mol/L sodium citrate buffer (pH 6.0) in a microwave. Endogenous peroxidase inhibition and blocking were performed. The paraffin sections were then incubated overnight with antibodies against CD68 (1:100), CD80 (1:100), CD163 (1:50), TGF-β1 (1:100), or RP S19 (1:100) after blocking with bovine serum albumin. Specific binding was detected by incubation with an HRP-conjugated secondary antibody, and positive staining was visualized with a DAB substrate kit. PBS was used instead of the primary antibody as a control. Images were captured using a digital camera connected to a microscope (BX512DP70; Olympus, Japan). The number of CD68/CD80/CD163-positive cells and the mean optical density of TGF-β1 and RP S19 were determined using ImageJ 1.53c software (Version: 2.1.0, National Institutes of Health, Maryland, United States).

The animals were killed under anesthesia at the end of experiment, and the middle lobes of right lung were excised, rinsed in saline and fixed with 4% paraformaldehyde in 0.01 M PBS (pH 7.4). The tissues were cut into blocks, embedded in paraffin, and sectioned to 5 μm sections. The sections were stained by hematoxylin and eosin (HE). The images of lung tissue were captured by a digital camera connected to a microscope (BX512DP70, Olympus, Tokyo, Japan).

Nissl staining was conducted on the sections using cresyl violet acetate as reported (Gu et al., 2018 (link)). The result was evaluated with a light microscope (BX512DP70; Olympus, Tokyo, Japan).

For immunohistochemistry (n = 4 for each group), coronal fresh frozen sections were sliced starting from the optic chiasma in 10 μm thick using a cryostat (CM1800, Leica, Bensheim, Germany). The slicing method was the same as above. The sections were incubated with mouse anti-CD31 (1:50, Thermo Fisher Scientific, MA1-80069, Waltham, MA, United States) diluted in PBS overnight at 4°C after blocking with bovine serum albumin. Then the samples were incubated with a biotinylated secondary antibody followed by avidin–biotin–peroxidase complex. Positive staining was visualized with diaminobenzidine. The images were captured by a digital camera connected to a microscope (BX512DP70, Olympus, Tokyo, Japan). Five fields were randomly selected for each rat. The number of open microvessels and microvessels with perivascular edema was analyzed with Image-Pro plus 5.0 software (IPP, Media Cybernetic, Bethesda, MD, United States). Five fields of the hippocampus and cortex region were randomly selected and examined separately for each animal (Gu et al., 2018 (link)). The microvessels that had the CD31-positive endothelium sticking together without any lumen were defined as closed microvessels. The percentage of microvessels with perivascular edema and closed microvessels in each field was calculated.

For immunohistochemistry, the sections were incubated with mouse anti-CD31 (1:50, Thermo Scientific, MA1-80069, Waltham, United States) diluted in PBS overnight at 4°C after blocking with bovine serum albumin. Then the samples were incubated with a biotinylated secondary antibody followed by avidin-biotin-peroxidase complex. Positive staining was visualized with diaminobenzidine. The CA1 region was magnified for 40 times and 200 times, captured by a digital camera connected to a microscope (BX512DP70, Olympus, Tokyo, Japan). And the number of open microvessels was analyzed with Image-Pro Plus 5.0 software (IPP, Media Cybernetic, Bethesda, MD, United States). Five fields of CA1 region were examined for each animal (Tian et al., 2013 (link)).

At the end of ischemia, hearts were cut from the middle one third between the apex and the ligation point and the lower part was fixed in 10% formalin, then prepared for paraffin sectioning. The paraffin sections (5 μm) were stained with hematoxylin and eosin (HE) and observed and photographed by a microscope equipped with a digital camera (BX512DP70, Olympus) (Lin et al., 2013 (link)).