Gb23303

Referring to the immunofluorescence experimental procedure described by Li et.al^{19 (link)}, RAW264.7 cells in different groups were fixed, permeated, blocked, and then incubated with NLRP3 (Cat No. MA5-23919; 1:200) and IL-18 (Cat No IPB0723; 1:200) primary antibodies at 4 °C overnight. The fluorescent secondary antibodies (GB23302, GB23303, Servicebio, 1:500) against the corresponding species were then incubated for 1 h and stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. After every step, the cells were thoroughly cleaned using phosphate-buffered saline (PBS) trice. Slides were observed under a fluorescence microscope.

The liver tissues were embedded in paraffin and sliced. The sample sections were deparaffinized and hydrated. After antigen retrieval, endogenous peroxidase was neutralized. ovine serum albumin was used to block antigen. The sections were incubated with CD31 primary (Abcam Cat# ab182981, Cambridgeshire, UK) and secondary antibody (Servicebio Cat: GB23303, Wuhan, China), the diaminobenzidine solution was added into the sections. After stained with hematoxylin, the sections were dehydrated and covered. The positive protein expression was examined by XSP-C204 (CIC, Beijing, China) and the images were analyzed by Image-Pro Plus 6.0.
Three high angiogenesis fields of each sample were chosen, and MVD (CD31) were calculated as numbers per field. The average number of three fields were considered as MVD of each sample. The operator was blinded to the group of sections.

Paraffin sections were deparaffinized, rehydrated, immersed in antigen retrieval solution, and autoclaved at 121° C for 10 min for antigen retrieval. Sections were incubated with Serum-Free Protein Block (Dako) and pretreated with 100% methanol containing 3% hydrogen peroxide. Then, sections were incubated with the AKAP8L antibody (36068; Signalway Antibody). Primary-stained sections were incubated with appropriate peroxidase-conjugated secondary antibodies (GB23303; Servicebio) and diaminobenzidine substrate.

The clinical samples were fixed in 10% neutral buffered formalin for 24 h. Paraffin was used for tissue embedding. Then tissue slides were well prepared and deparaffinized using dimethylbenzene, anhydrous ethanol, 85% ethanol, 75% ethanol, and distilled water orderly. The container with ethylene diamine tetraacetic acid (EDTA) antigen repair buffer (PH 9.0, Servicebio, G1203) for MYD88 or citric acid tissue antigen repair solution (PH 6.0, Servicebio, G1202) for CD68 and CD163 in the microwave oven was used correspondingly to repair the antigen of the slides using median fire to boiling for 8 min, keeping warm for 8 min and median-low fire for 7 min consecutively. Peroxidase was blocked using the 3% H₂O₂ for 25 min. Then we blocked the antigen using goat serum (Servicebio, G5001) for 30 min. We used MYD88 (1:20, CST, 4283S),CD68 (1:200, Servicebio, GB14043) and CD163 (1:600, Servicebio, GB13340) antibody overnight at 4°C to stain the slides, among which the two adjacent slides were stained with CD68 and CD163 separately. Then the slides were incubated with secondary antibodies (1:200, Servicebio, GB23303) for MYD88 and CD163 or secondary antibodies (1:200, Servicebio, GB23301) for CD68 50 min at room temperature. After adding DAB (DAKO, K5007) and hematoxylin (Servicebio, G1004) staining, slides were covered and observed by microscope (Grundium OCUS).

TGF-β1, α-sma, and COL1A1 immunohistochemistry (IHC) staining were performed after the samples were embedded in paraffin and sliced. Sections were placed in xylene for 3 times of 15 min each, absolute ethanol for 3 times, and subsequently rinsed with deionized water. The samples underwent antigen retrieval, and then were placed in 3% methanol hydrogen peroxide for 25 min, rinsed with deionized water, and placed in phosphate buffered saline (PBS) for 3 times. The slides were incubated with rabbit TGF-β1 (1:500 dilution, Servicebio, GB111876), α-sma (1:5000 dilution, Servicebio, GB111364), or COL1A1 (1:5000 dilution, Servicebio, GB11022-3) antibody at 4 °C overnight. The slides were washed in PBS for 3 times and incubated with the secondary antisera (1:200 dilution, Servicebio, GB23303) for 50 min at 37 °C. The slides were washed in PBS for 3 times, 3,3′-diaminobenzidine tetrahydrochloride (DAB) was applied, and rinsed in PBS and deionized water. The slides were stained with hematoxylin for 3 min as a counterstain, and washed in tap water. The pathology slices were photographed by an electron microscope and analyzed by Image J software.

Total proteins were extracted from cells with lysis buffer and were incubated for 15 min at 95°C. Then, proteins were subjected by 10% SDS-PAGE and then transferred to PVDF (polyvinylidene fluoride) membranes (0.45 μm pore size; Millipore, MA, USA). The membranes were blocked with quick blocking buffer (P0252; Beyotime, China) for 10 min and then incubated with the anti-TLR4 (1 : 1,000; sc-293072; Santa Cruz), anti-SHH (1 : 1,000; 20697-1-AP; Proteintech), anti-SMO (1 : 2,000; ab266423; Abcam), anti-Gli1 (1 : 1,000; A14675; ABclonal), anti-p16 (1 : 1,000; A11651; ABclonal), anti-p53 (1 : 1,000; A19585; ABclonal), anti-MMP9 (1 : 1,000; 10375-2-AP; Proteintech), or anti-β- actin (1 : 4,000; 20536-1-AP; Proteintech) overnight at 4°C. The membranes were washed with TBST three times and then incubated with a secondary anti-rabbit antibody (1 : 4,000; GB23303; Servicebio) or anti-mouse antibody (1 : 4,000; GB23301; Servicebio) for 1 h. Finally, the membranes were visualized in chemiluminescence method (WVKLS0500; Millipore). All experiments were performed three times.