Abs50012

IHC was performed in ESCC specimen of paratumour tissue, high grade dysplasia, intramucosal ESCC and progressive ESCC. Samples were used for staining of LUM (Cat#ab168348; Abcam) NCL (Cat#ab129200; Abcam) and MDK (Cat#ab52637; Abcam) antibodies. All the staining process was carried out on the IHC/ISH System (BenchMark GX, Roche) following the manufacturer's instruction. Multiplexed IHC (mIHC) was performed using a multiple fluorescent immunohistochemical staining kit (abs50012; Absin, Shanghai, China) according to the manufacturer's instructions.¹⁹ Sections were observed using an optical microscope (BX53; Olympus, Tokyo, Japan)

Multiplex fluorescence according to the manufacturer’s instructions (Absin, abs50012).

Tissue sections were prepared as described in the previous section. After deparaffinization and rehydration through ethanol gradients (95%, 80%, 70%), sections underwent an antigen repair process in sodium citrate buffer using microwave thermal repair and endogenous peroxidase blocking in 0.3% hydrogen peroxide. After nonspecific reactions were blocked with 5% goat serum at 20℃ for 10 min. Sections were first incubated with anti-COLIII primary antibody (GB111629, Servicebio, Wuhan China) overnight at 4 °C, then incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody, and fluorescent dye was ligated to it according to the manufacturer’s instructions (abs50012, Absin, Shanghai, China). After washing with PBST 3 times, sections were further incubated with the second primary antibody (anti-SOX5 (A6985, Abclonal, Wuhan, China), anti-NFATC2 (A3107, Abclonal), anti-NPAS2 (A16930, Abclonal)) at room temperature for 1 h. The procedures were repeated starting from the second antibody incubation. After all the procedures were completed, the slides were washed in PBST, counterstained with DAPI, and sealed with anti-fluorescence quenching sealing tablets. The stained tissue sections were examined using a Leica DMI8 microscope (Leica, Germany).

Immunofluorescence staining was performed by using a multiplex immunofluorescence staining kit (abs50012, Absin) as previously described.⁴¹ The tissue sections were dewaxed in xylene, dehydrated in alcohol, repaired by microwave and blocked with 5% goat serum. The sections were incubated with primary antibody anti‐iba1 rabbit monoclonal (1:2000, Abcam) at room temperature for 1 h. The secondary antibody was incubated for 10 min and incubated with monochromatic fluorescent dyes 520 for 10 min, and then, microwave repair was cooled to room temperature. The above steps were repeated to complete the staining of anti‐METTL3 rabbit monoclonal (1:500, Abcam). We used 570 as a fluorescent dye for METTL3. Finally, the sections were stained with DAPI and anti‐fluorescence quencher seal. Images of stained sections were obtained by confocal microscope (Nikon A1+). The number of METTL3 or iba1 positive cells was calculated using Image‐Pro Plus 6.0.

ACAN, MMP13, β-catenin, iNOS and CD206 in pathological sections were examined by immunohistochemistry. Paraffin sections of joints were dewaxed, rehydrated, pretreated with pepsin at 37 °C for 30 min, and then incubated with 3% H₂O₂ in methanol solution. After rinsing with PBS, the slices were blocked with BSA at room temperature for 1 hour and then incubated overnight with primary antibodies at 4 °C. Then, the slices were incubated with the secondary antibody provided in the HRP polymer anti-rabbit IHC kit (Kit 5005; MaxVision, Shenzhen, China) for 15 min and stained with substrate from the DAB Plus kit (DAB-2031) for 10 min. Histological images of the knee joint were obtained after staining with safranin O and fast green. We performed immunofluorescence using a multicolored immunofluorescence kit (abs50012, absin) and observed the cells using a microscope from Zeiss.

For immunofluorescence staining, we utilized a multiplex immunofluorescence staining kit (abs50012, absin, Shanghai, China) and followed the instructions provided by the manufacturer. Antibodies against NDRG1 (1:500, T57079S, Abmart) and CD206 (1:500, TD4149S, Abmart) were incubated at room temperature for one hour. Subsequently, the slides were incubated with anti-rabbit/mouse IgG conjugated with HRP for 15 minutes at room temperature, followed by incubation with fluorophore-conjugated tyramine molecules (PPD 650, PPD 570, or PPD 520) for 15 minutes. Finally, the nuclei were stained using DAPI.

Formalin-fixed paraffin-embedded sections of PA specimens were collected at the Hospital of Stomatology, Sun Yat-sen University. To detect the protein expression levels of marker genes, the PA paraffin sections were stained four-color-multilabeled immunofluorescence staining kit (Absin, Cat#abs50012) according to the manuffacture’s protocols. Briefly, the sections were incubated in 3%H₂O₂ for 10 mins for the first time. Every time the sections were incubated with antibodies against CD36 (ab17044, Abcam), Pan-keratin (PCK) (26411-AP, Proteintech), ACTA2 (ab220179, Abcam). After each incubation of the primary antibody, heat-induced epitope recovery and 5% BSA blocking were performed. The HRP conjugate and three wavelengths (520,570 and 650 nm) were utilized to attach the different primary antibodies. Then, the slides were counterstained with DAPI for nuclear visualization, and subsequently coverslipped with fluorescence quenching mounting medium. Images were acquired using a fluorescence microscope. The organoid immunofluorescence staining was performed as described previously with antibodies against CD36 and PCK^{68 (link),69 (link)}. All primary antibodies were used at a dilution of 1:200.