Zli 9018

Strict serial 4 μm sections were cut from formalin-fixed paraffin-embedded tissue blocks. The detection of Keratin 7-positive cells was performed via staining the sections with a primary antibody against Keratin 7 using SP kit (SP-9002, ZSGB-BIO) according to the manufacturer’s instructions. Stains were detected using anti-immunoglobulin-coupled horseradish peroxidase with 3,3′-diaminobenzidine (DAB, ZLI-9018, ZSGB-BIO) as substrate. Nuclear counterstaining was performed with Cole hematoxylin (G1140, Solarbio).

Tissues were cut into 3-μm-thick sections and incubated for 1 h with 3% hydrogen peroxide to eliminate endogenous peroxidase activity after deparaffinisation and rehydration. After antigen retrieval, sections were incubated with BDH2 antibody (1:1000, HPA004428, Sigma, St. Louis, MO, USA) at 4 °C overnight, then secondary antibody (ZB-2305, ZSGB-BIO, Beijing) for 1 h at room temperature. A 3,3′-diaminobenzidine (DAB) reagent (ZLI-9018, ZSGB-BIO, Beijing) was used for peroxidase reaction, and haematoxylin for counterstaining. Images were acquired under a microscope (C-5050, Olympus, Japan). The results were independently evaluated in a blinded manner by two pathologists. The intensity of BDH2 staining was scored as described.^{20 (link)}

The immunohistochemical staining was performed as described by Shin et al. (2019) (link) with slight modifications (Shin et al., 2019 (link)). After being fixed in 4% paraformaldehyde for 24 h, the brains were embedded in paraffin and then cut at a thickness of 5 μm. The sections for staining were deparaffinized and washed. Following, they were heated in 0.01 M sodium citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxide activity was quenched with 3% hydrogen peroxide. Sections were incubated with primary antibodies against Aβ1–42 (Abcam, ab10148, 1:200) overnight at 4°C, followed by anti-rabbit IgG (ZSGB-BIO, PV-9001). Then sections were incubated with horseradish enzyme labeled streptomycin working solution at room temperature for 30 min. Finally, color was developed with 3,3′-diaminobenzidine (ZSGB-BIO, ZLI-9018), and then it was counterstained with hematoxylin. After gradient dehydration with alcohol, the slices were sealed with neutral resin. The images were captured. And 6 visual fields per slices were being counted in a blind manner.

Mice liver tissue samples were fixed overnight in 4% formalin, processed for paraffin embedding, and sectioned (5 µm per section). Tissue sections were stained with hematoxylin and eosin (H&E) and photographed by light microscopy. Areas of necrotic or imminent necrosis of tissue were identified by the presence of eosinopenia, loss of cell structure, vacuolization, cell rupture, or nucleolysis. To facilitate the analysis, necrotic areas with all features and areas about to undergo necrosis without nucleolysis but with most features were included in the quantitative analysis of necrosis. Immunohistochemistry staining was performed to analyze the expression profiles of specific proteins in liver tissue. Briefly, slides were de‐paraffinized, rehydrated, and boiled in a microwave for 10–12 min in 10 × 10⁻³m citrate buffer or Tris‐EDTA buffer (pH 9.0). After incubation with 2% bovine serum albumin for 30 min at room temperature, liver sections were incubated with primary antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)‐conjugated secondary antibodies (ZLI‐9018, ZSGB‐BIO) for 2 h at room temperature. Specimens were washed three times then developed with the DAB substrate kit (ZLI‐9018, ZSGB‐BIO) and counterstained with hematoxylin. Antibodies used in this study are summarized in Key Resources Table.

The protein expression levels of PRC1 and RACGAP1 were detected by immunohistochemistry in paraffin-embedded sections of 5-μm-thick liver cancer tissue and adjacent normal tissue, as described (Chen et al., 2016 (link)). Paraffin-embedded sections were first taken for dewaxing and antigen repair, and then the samples were incubated with PRC1 or RACGAP1 antibodies (Proteintech, #15617-1-AP, #13739-1-AP) at 4°C overnight. After incubation, the slides were cleaned with phosphate-buffered saline with Tween 20 (PBST) and incubated with HRP-conjugated secondary antibodies (Proteintech, #SA00001-2) at room temperature for 1 h. After PBST cleaning, the slides were colored with DAB (ZSGB-BIO, #ZLI-9018). Then the slides were stained with hematoxylin staining solution (solarbio, #G1080), dehydrated, and sealed with a neutral gum. Finally, the slides were observed using the SLIDEVIEW VS200 slide scanner (Olympus).

Paraffin-embedded kidney sections were incubated at 60 °C for 60 min, deparaffined with xylene three times for 15 min each, hydrated with gradient ethanol solution for 5 min each, and finally immersed in deionized water. Following a 20 min incubation at 95 °C with an antigen retrieval solution for antigen retrieval, the sections were let to cool naturally to room temperature. The endogenous peroxidase activity was quenched by incubation with 3% H₂O₂ at room temperature for 10 min. The sections were then blocked with goat serum (ZLI-9056, ZSBIO company, Beijing, China) at 37 °C for 30 min, followed by incubation with anti-nephrin (1:2000, ab216341, Abcam), anti-podocin (1:1000, ab50339, Abcam) and anti-cleaved caspase-3 (1:400, 19677-1-AP, Proteintech, Wuhan, China) antibodies overnight at 4 °C. The sections were then washed in phosphate-buffered saline (PBS), treated for 20 min at room temperature with horseradish peroxidase-conjugated anti-rabbit secondary antibody (PV-9001, ZSBIO business), and colour development was performed by incubating with diaminobenzidine (DAB, ZLI-9018, ZSBIO company). Finally, the nuclei were stained with haematoxylin. At least five randomly chosen fields for each sample were evaluated under the microscope and analysed with Image-Pro Plus 6.0 software.