Goat serum

Each group was fixed in 4% PFA for at least 30 min. Then the cells were collected by centrifuging at 1500 rpm for 5 min after cracked the composite hydrogels by using lysate (8.09 g sodium citrate, 4.39 g sodium chloride, 2.92 g EDTA, 500 ml deionized water) [17 (link)]. Washed the cells for 3 min 2 times with PBS and blocked for 30 min with 0.3% Triton X-100%. Then, the cells were washed with PBS once more. After permeabilization with 5% goat serum (Zsbio, China) at 37 °C for 30 min, the cells were incubated with primary antibodies overnight at 4 °C. After the cells were incubated with second antibodies for 2 h at room temperature, they were washed twice with PBS. Finally, the cells were incubated for 10 min with DAPI (1:300, Beyotime) as a nuclear stain. Images were scanned with fluorescence microscopy (Leica BMI4000, Germany) and a confocal microscope (Leica, TCSSP8, Germany). The antibodies used were as follows: keratin-8 (K8) (rabbit, 1:200, Abcam), K14 (mouse, 1:200, Abcam), K19 (rabbit, 1:200, Abcam), ATP1a1 (rabbit, 1:200, Abcam), estrogen receptor-α (ER-α) (rabbit, 1:200, Abcam), goat anti-rabbit Alexa Flour 488 (1:300, Beyotime), and goat anti-mouse Alexa Flour 488 (1:300, Beyotime), goat anti-rabbit Alexa Flour 594 (1:300, Beyotime), and goat anti-mouse Alexa Flour 594 (1:300, Beyotime).

Immunofluorescence analysis was performed as previously described 7. Briefly, the cells or EC matrix was fixed with 4% paraformaldehyde for 20 min. at room temperature, followed by permeabilization with 0.3% Triton X‐100 in PBS for 5 min. The cells were rinsed and blocked with either 10% goat serum (Zsgb‐Bio, Beijing, China) or 5% BSA (Invitrogen) for 60 min. at room temperature. The cells were then incubated with the primary antibodies, which are listed in Table S1, at 4°C overnight. Following three 5‐min. washes in PBS with gentle agitation, an Alexa Fluor‐conjugated secondary antibody (Invitrogen) at 1:500 was added, and the samples were incubated for 1 hr at 37°C. The nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma‐Aldrich).

Tumor slides and the CRC cohort were deparaffinized and rehydrated, followed by antigen retrieval for 3 min (high temperature and high pressure). The slides were then incubated with goat serum (Zsbio, Beijing, China; room temperature, 15 min) and washed with PBS three times. The slides were then incubated overnight with primary antibodies against ZNF575 (1:100, bs-13588R; Bioss, Shanghai, China) and p53 (1:500, 21891-1-AP; Proteintech, Wuhan, China) at 4°C. After incubation with secondary antibodies (Zsbio, Beijing, China; room temperature, 2 h), slides were stained with 3,3′-diaminobenzidine (Maixin, Fuzhou, China). Hematoxylin was used to stain cell nuclei. Apoptotic cells in tumor tissues were detected using the TUNEL assay (Beyotime, Beijing, China) following the manufacturer’s instructions. Cell nuclei were stained with DAPI (Beyotime, Beijing, China). All the slides were photographed using an Olympus BX600 microscope (Olympus, Tokyo, Japan).

BV2 microglia were grown on coverslips in 24-well plates. After fixation in 4% paraformaldehyde for 15 min, the cells were blocked with goat serum (ZsBio, China) for 20 min at room temperature and then incubated at 37 °C for 1 h with rabbit anti-mouse Arg1 (Millipore, USA), rabbit anti-human CD206 (Santa Cruz, USA) and mouse anti-mouse phospho-AKT (CST, USA). After the slides were washed three times, they were incubated at 37 °C for 1 h in the dark with a secondary fluorescent chicken anti-rabbit IgG (H + L) CF633 (Sigma-Aldrich, USA) and rabbit anti-mouse AlexaFluor 488 (Lifetech, USA). After the slides were washed, the cells were stained with DAPI (Beyotime Biotechnology, China) for 15 min at 37 °C, and then the slides were mounted with an anti-fluorescence quenching agent. Images were visualized using an LSM 780 confocal laser scanning microscope (Carl Zeiss GmbH, Germany).

The tissue array (Outdo Biotech, Shanghai, China) was applied for immunohistochemistry (IHC) staining to detect CHD5 expression in 90 RCC patients. Antigen unmasking was performed in citrate buffer by microwaving after deparaffinization and rehydration, followed by inactivation of endogenous peroxidase with 0.3% H₂O₂. Sections were blocked with goat serum (ZSGB-BIO, Beijing, China) and incubated with CHD5 (Cell Signaling Technology, Beverly, USA; 1 : 100 dilution) overnight at 4°C. Subsequently, sections were probed with anti-rabbit antibody and avidin-biotin peroxidase at room temperature and visualized using diaminobenzidine before counterstaining with hematoxylin. The percentage of positive cells (PP) were graded as follows: 0 (<1%), 1 (1-10%), 2 (11-50%), and 3 (>50%). Staining intensity (SI) was defined as follows: no staining, weak staining, moderate staining, and strong staining, corresponding to 0-3 points. PP multiplied by SI was identified as immunoreactivity scoring (IRS). IRS 0–1 was defined as low expression, and >1 was defined as high expression [19 (link)].

The cells were seeded to a 24-well plate with 20,000 cells/well and cultured overnight in the incubator. Then they were cultured in the incubator for 48 h after the medium containing DMSO or BD was added to each well. After incubated with BrdU (Abcam, United States, 30 μg/ml) for 45 min, the cells were washed with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) for 15 min. Subsequently, 200 μl 2 M hydrochloric acid was added to each well at 37°C for 20 min. After washing with PBS for three times, 10% goat serum containing 0.5% Triton X-100 (ZSGB-Bio, Beijing, China) was used for blocking at room temperature (RT) for 2 h. The cells were incubated with anti-BrdU monoclonal rat first antibody (1: 1,000) overnight at 4°C. The samples were incubated at RT for 2 h with Alexa FluorR^®488 goat anti-mouse IgG second antibody (HG L; 1: 10,000, antioxidant). The nuclei were stained with DAPI (1: 1,000). The percentage of BrdU staining was calculated from at least five microscopic visual fields.

Tumor specimens were fixed in 10% formaldehyde solution and embedded in paraffin. The paraffin-embedded tissues derived from clinical specimens of bladder cancer were sectioned at 4 μm thickness and mounted on glass slides in sequence. After being baked at 60°C for 2 h, the sections were then deparaffinized in xylene, rehydrated through grade ethanol, and eliminated the endogenous peroxidase activity in 3% hydrogen peroxide. For antigen retrieval, the sections were submerged in citrate or EDTA buffer and boiled in the pressure cooker for 2 min. To block nonspecific background, goat serum (ZSGB-BIO, China) was applied. The sections were incubated overnight at 4°C with specific primary antibodies against LAG3, VEGF, and TNFSF14 and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibodies. After a brown stain was generated with DAB conjugated by horseradish peroxidase, slides were counterstained with hematoxylin, dehydrated, and covered with coverslips. All of the slides were assessed by two urological pathologists.