3 3 diaminobenzidine dab substrate kit

After deparaffinization and antigen retrieval, the tissue sections were incubated with anti-TPM2 antibody (1:100, Proteintech, 28587-1-AP), anti-YAP1 antibody (1:100, Proteintech, 13584-1-AP), or anti-Ki67 (1:100, Proteintech, 27309-1-AP) antibody overnight at 4 °C. Then, a peroxidase-conjugated secondary antibody was used to detect antigen–antibody complexes. After that, a color reaction was conducted using a 3,3′-diaminobenzidine (DAB) substrate kit (ZsBio). ImageJ software was used for IHC quantification. To perform immunofluorescence staining, PCa cells were seeded on coverslips and fixed in 4% PFA. Cells were then washed and treated with 0.25% Triton X-100 for 15 min. After blocking by 5% donkey serum at room temperature for 30 min, multicolor immunofluorescence staining was performed by Treble-Fluorescence immunohistochemical mouse/rabbit kit (RS0036; Immunoway) according to the manufacturer’s protocol.

ccRCC tissue sections were deparaffinized and the antigen was retrieved, followed by blocking with 5% bovine serum albumin. Next, ccRCC tissue sections were incubated with KIF23 primary antibody (1:100, Proteintech, 28587-1-AP) at 4°C overnight, a peroxidase-conjugated secondary antibody was used to detect antigen-antibody complexes. Thereafter, a color reaction was conducted using a 3,3′-Diaminobenzidine (DAB) substrate kit (ZsBio). For immunofluorescence staining, ccRCC cells were seeded on coverslips and fixed in 4% PFA. Then, cells were washed and treated with 0.25% Triton X-100 for 15 min. After blocked by 5% donkey serum at room temperature for 30 min, cells were incubated with β-catenin (1:100, Cell Signaling Technology, 8480) at 4°C overnight. Alexa Fluor 594 conjugated secondary antibody (1:500; Yeasen) was used to detect antigen-antibody complexes. 4′,6-Diamidino-2-phenylindole (DAPI) was used for the staining of nucleus.

Antigens were recovered from the deparaffinized tissue sections and blocked with 5% bovine serum albumin. Tissue sections were incubated with primary and peroxidase-conjugated secondary antibodies overnight at 4 °C to detect antigen-antibody complexes. Thereafter, a color reaction was performed using a 3,3′-diaminobenzidine (DAB) substrate kit (ZsBio). Briefly, RCC cells were plated on coverslips and fixed with 4% PFA for immunofluorescence staining. The cells were treated with 0.25% Triton X-100 for 15 min and blocked with 5% donkey serum. The tissue sections were mixed with primary antibody overnight at 4 °C followed by multicolor immunofluorescence staining using the Treble-Fluorescence Immunohistochemistry Mouse/Rabbit Kit (Immunoway) according to the manufacturer’s protocol. Alexa 488 conjugated phalloidin (Beyotime) was used to stain F-actin.

For histochemical analysis of the brain, mice were anesthetized and perfused transcardially with 0.01 M of phosphate-buffered saline (PBS) (pH 7.4) and 4% paraformaldehyde in 0.01 M of PBS. The brain was removed and then switched to 30% sucrose/0.1 M of PBS. After being frozen in isopentane, the brain tissues were cut in serial, 30-μm-thick horizontal sections in a cryostat slicer (CM1900, Leica, Germany). The brain sections were exposed to 3% H₂O₂ for 10 min, blocked by 5% normal goat serum in PBS for 60 min, and then incubated with AT8 (1:200, an antibody against PHF tau phosphorylated at Ser202/Thr205; Thermo Fisher Scientific, United States) at 4°C overnight. The sections were incubated with a goat anti-mouse non-biotin detection system (Zsbio, China) at 37°C and visualized using 3,3′-diaminobenzidine (DAB) substrate kit (Zsbio, China). Sections were counterstained with hematoxylin, dehydrated in ascending concentrations of ethanol, and coverslipped. Slices were imaged using the Olympus microscope (BX51, Olympus, Japan) and Pixera TWAIN View Finder Pro system. Images were quantified using IHC Profiler and threshold in ImageJ software.

Briefly, 3-µm paraffin-embedded tissue sections were deparaffinized, rehydrated, treated with 0.3% hydrogen peroxide, and processed for antigen retrieval with heat induction for approximately 10 min^{26 (link)}. Primary antibodies to the following proteins were used for IHC staining: CD66b (1:100, Proteintech Group, Chicago, IL, USA), neutrophil elastase (NE) (1:100, Abcam, Cambridge, Cambridgeshire, United Kingdom), and IL-8 (1:100, Affinity Biosciences, Cincinnati, OH, USA). Tissues were then incubated with peroxidase conjugated goat anti-mouse/rabbit IgG secondary antibody (ZSGB-bio, Beijing, China). The slides were finally stained with 3,3´-diaminoben-zidine (DAB substrate kit, ZSGB-bio, Beijing, China) and counterstained with hematoxylin. In negative controls, the primary antibody was replaced with phosphate buffered saline. The specimens were analyzed under a light microscope (Nikon, Tokyo, Japan) by pathologists.
Semiquantitative analysis was performed according to IHC scores^{24 (link),27 (link)}. The proportion of stained cell counts per field was scored as 0, 1, 2, and 3 if there was no positive staining, < 10% positive staining, 10%–30% positive staining, and > 30% positive staining, respectively. IHC scoring of cytokine expression was classified into 4 categories (0, 1, 2, and 3) according to the staining intensity (none, weak, moderate, and strong).