Coralite488 conjugated affinipure goat anti rabbit igg h l

The cultured cells were fixed with 4% paraformaldehyde at room temperature for 20 min, then permeabilized with 0.3% Triton X-100 for 15 min, blocked with 3% BSA for 30 min. After discarding the block solution, the samples were incubated with Ki67 (1:300, ab15580, abcam, United Kingdom) overnight, and then incubated with CoraLite 488-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00013-2, proteintech, China) for 1.5 h. The nucleus was stained with DAPI (C1002, Beyotime, China) and the cytoskeleton was stained with Actin-Tracker Red 594 (F-Actin, C2205S, Beyotime, China). The number of the cell proliferation marker Ki67 was observed and recorded with a fluorescence inverted microscope (Nikon, Japan) at a wavelength of 550 nm.

PK-15 cells were seeded on coverslips in six-well cell culture plates and transfected with the corresponding plasmids for 36h. Then, the cells were fixed with 4°C precooled anhydrous methanol at room temperature for 10min after washing the coverslips three times with PBS. The cells were blocked with 5% bovine serum albumin for 45min, and then a rabbit polyclonal anti-LC3B antibody, a rabbit polyclonal anti-Myc-tag antibody, or a mouse monoclonal anti-Flag-Tag antibody was added to the cells and incubated at 4°C overnight. Next, after being washed three times with PBS, the samples were incubated with the secondary antibody CoraLite488-conjugated AffiniPure goat anti-rabbit IgG (H+L; Proteintech) or Cy3-labeled goat anti-mouse IgG (H+L; Beyotime, China) at 37°C in the dark for 2h. After washing three times with cold PBS, the cell nuclei were stained with 2.5μg/ml DAPI (Sigma, D9542) in PBS for 15min at 37°C. The samples were observed using a confocal scanning laser microscope (Zeiss LSM880). PK-15 cells were transiently transfected with mRFP-eGFP-LC3 constructs to monitor autophagic flux. After 36h, the cells were treated with the indicated compounds (The autolysosome inhibitor bafilomycin A1, BAF) for 12h. All samples were analyzed using a laser scanning confocal microscope.

We deparaffinized the sections to water and placed in xylene for 20 min, which were performed three times. Slices were put in 100, 95, 85, and 75% ethanol in sequence for 5 min at each level. Slices were washed with distilled water for 5 min. The slices were immersed in citrate buffer (pH 6.0) and boiled in an electric furnace or microwave oven. After being cooled, slices were washed with 0.01 M of PBS (pH 7.2 ~ 7.6) for 3 min, which were performed three times. Slices were place in sodium borohydride solution at room temperature for 30 min. The sections were placed in Sudan black dye solution at room temperature for 5 min. Slices were blocked with 10% normal serum/5% bovine serum albumin (BSA) for 60 min. Slices were placed in appropriate first antibody, cAMP (1:50, rabbit, ab76238, Abcam, UK), MUC2 (1:50, rabbit, ab76774, Abcam, UK), Reg3γ (1:50, rabbit, ab233480, Abcam, UK), β-defensin (HBD-2) (1:50, rabbit, bs-1296r, Bioss, China), Claudin-1 (1:50, rabbit, 13050-1-AP, PTG), and ZO-1 (1:50, rabbit, 21773-1-AP, PTG), overnight at 4°C. Slices were incubated with CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (SA00013-2, Proteintech, USA) and incubated at 37°C for 90 min. Slices were stained in the nucleus with DAPI (Wellbio, China) working solution at 37°C for 10 min. Slices were stored in the dark or observed under a fluorescence microscope.

For immunocytochemistry, cells were seeded on poly-D-lysine precoated cell climbing films (Shanghai wohong Biotechnology Co., Ltd) in 12-well culture plate. The medium was removed and the cells were washed with PBS three times for 5 min each and then fixed with 4% paraformaldehyde for 15 min at room temperature. After washing with PBS three times for 5 min each, the fixed cells were blocked by using 0.3% Triton X-100/PBS for 1 h. Cells were incubated with the primary antibody, HIF-1α (Proteintech: 20960-1-AP), PKM2 (Proteintech: 60268-1-Ig), GLUT-1 (Proteintech: 66290-1-Ig), LDH-A (Proteintech: 19987-1-AP) diluted in 1% BSA in PBS overnight at 4°C. After washing three times with PBS, cells were incubated for 1.5 h with CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (Proteintech: SA00013-2) or CoraLite594-conjugated Goat Anti-Mouse IgG (H + L) (Proteintech: SA00013-3) secondary antibodies. The unbound secondary antibody was removed with three washes of PBS for 5 min each. Next, the samples were counterstained with DAPI (Beyotime Biotechnology). Samples were visualized on fluorescence microscopes (Nikon, Japan). The intensity was quantified using ImageJ software.

The HUVECs were treated as we described previously.34 Cell was incubated with FITC‐phalloidin (Cat. No: P5282; Sigma, Germany), VE‐Cadherin Rabbit antibody (1:400, #2500; Cell Signal Technology, USA) and Claudin‐5 Rabbit antibody (1:200, Cat. No. ab15106; Abcam, Cambridge, MA, USA) overnight at 4°C. After washing three times, the cells were incubated with CoraLite594‐conjugated Goat Anti‐Rabbit IgG(H+L) (1:200, Cat. No. SA00013‐4; Proteintech) and CoraLite488–conjugated Affinipure Goat Anti‐Rabbit IgG(H+L) (1:200, Cat. No. SA00013‐2; Proteintech) for 1 hour. The HUVECs were again washed with PBS and incubated with DAPI (1:2000, Cat. No. D9564; Sigma‐Aldrich) for 10 minutes. Finally, the cells were washed three times again in PBS and observed under a confocal microscope (Olympus FV‐1000).

To assess Ki67 expression in the mouse tumors, The sections were immersed in a solution of sodium borohydride solution, soaked in 75% ethanol solution, and then placed in Sudan Black staining solution. The sections were incubated in a 5% BSA solution. Appropriate dilutions of primary antibody Ki67 (ab16667, 1:100, Abcam) were added dropwise overnight at 4℃. 50–100 µL CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00013-2, Proteintech) fluorescent antibody was incubated. DAPI was used to stain nuclei. The sections were sealed using buffered glycerol and subsequently examined using a fluorescence microscope.

Iso (purity ≥ 98%) was obtained from Pharmaceutical Technology Co., Ltd. (Jiangsu, China) and BaP was purchased from Sigma (St. Louis, MO, USA). Caspase-1 activity assay kit and 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA) were obtained from Beyotime biotechnology Co., Ltd. (Shanghai, China). RPMI-1640 medium, BCA protein kit, and penicillin–streptomycin solution were from Thermo Fisher (Shanghai, China).
TritonX-100, DAPI-Fluoromount-G, Bovine Serum Albumin (BSA), and CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) were from Proteintech Group, Inc (Rosemont, IL, USA).
Polyclonal antibodies specific to NLRP3 (GTX106313), ASC (10500-1-AP), NF-κΒ (10745-1-AP), GSDMD (20770-1-AP), IL-1β (16806-1-AP) and IL-18 (SC-6179) were purchased from Santa Cruz Biotechnology (Dallas, Texas, United States). GAPDH (BA2913) was obtained from Bioworld Technology, Inc. (Louis Park, MN, USA). Caspase-1 (2225) was purchased from Cell Signaling Technology (Shanghai, China).
Specific inhibitors MCC950, N-acetylcysteine (NAC), and Z-YVAD-FMK were obtained from Beyotime biotechnology Co., Ltd. (Shanghai, China). Ammonium pyrrolidinedithiocarbamate (PDTC) and Mito-TEMPO were obtained from Sigma (St. Louis, MO, USA). All other reagents were dissolved in water or DMSO, and then diluted with fresh RPMI-1640 medium.