Bx40 light microscope

Slides of bone tissue were allowed to warm to room temperature, then fixed in 10% neutral-buffered formalin. Membranes in the tissue were permeabilized using 0.25% Triton X-100 (catalog no. 9002-93-1; Sigma-Aldrich, Darmstadt, Germany). Endogenous peroxidase activity was quenched using 3% hydrogen peroxide (Solarbio, Beijing, China), and antigens were retrieved using citrate buffer on a hot plate at 85°C. Non-specific reactivity on slides was blocked by incubating them in 5% bovine serum albumin at room temperature, then primary antibody (diluted 1:100) was added for overnight incubation at 4°C. Next, secondary antibody was applied for 60 min at room temperature. Slides were rinsed with 1 × Tris-buffered saline containing 0.5% Tween-20 (TBST), incubated for 30 min with avidin–biotin peroxidase (diluted 1:200), rinsed again in 1× TBST, then developed using 3,3-diaminobenzidine (Vector Laboratories, Burlingame, CA, United States). Slides were examined using an Olympus BX40 light microscope and photographed at ×20 magnification.

Heart allografts were fixed with 4% paraformaldehyde and their paraffin-embedded sections were stained using H & E (hematoxylin and eosin) stain. Tissue injury by rejection was semiquantitatively assessed according to a previous study [24 (link)]. Immunohistochemical staining was also performed using rabbit anti-CD3 (DAKO, Glostrup, Denmark) or anti-mouse F4/80 (clone BM8, Thermo Fisher Scientific), followed by a Dako Real Envision HRP kit (DAKO). The stained slides were assessed using an Olympus BX40 light microscope (Olympus, Japan), and images were analyzed with Image-Pro 5.1 software (Media Cybernetics, Rockville, MD, USA). All histological analyses were performed by two independent researchers blinded to the treatment group.

HCT116 cells with 5 × 10² cells/well suspended in RPMI-1640 medium were seeded into six-well plates and cultured in a 5% CO₂ incubator at 37°C for 14 days. Subsequently, these cells were subjected to fixation in 70% ethanol at room temperature for 15 min and staining in 0.05% crystal violet for 20 min at 37°C. The number of colonies formed (>50 cells/colony) was calculated by counting with an Olympus BX40 light microscope (Olympus Corporation) [26 (link)].

Paraffin‐embedded synovial tissues were also analysed using immunohistochemistry. Sections were deparaffinized in xylene, rehydrated in ethanol, microwaved in citrate buffer to retrieve antigens, incubated in 3% H₂O₂ for 10 min followed by goat serum for 60 min at room temperature, and then incubated overnight at 4°C with antibodies against NLRP3 (1:100 dilution; catalogue no. MAB7578, R&D, Minneapolis, MN, USA), caspase‐1 (1:100; MAB62154, R&D) or GSDMD‐N (1:500; 36,425 s, CST,). Next, sections were incubated for 30 min at room temperature with horseradish peroxidase‐linked goat secondary antibodies against rat IgG, mouse IgG or rabbit IgG (CST), followed by treatment with 3,3′‐diaminobenzidine (catalogue no. SK4100, Vector Labs,) and Mayer's haematoxylin. Stained sections were viewed under a BX40 light microscope (Olympus).

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to study the DNA fragmentation or apoptosis detection. TUNEL assay was performed on 4–5 µm thick sections as per the kit manual. Formalin-fixed tumor tissues were embedded in paraffin and sectioned (4–5 mm thick). DeadEndTM Colorimetric Apoptosis Detection System (Promega, Madison, WI) allows the recognition of apoptotic nuclei in paraffin-embedded tissue sections fixed on slides according to the manufacturer's protocol. Briefly, the equilibration buffer was added to slides and incubated for 10 minutes followed by 10-minute incubation in 20 mg/ml proteinase K solution. The sections were washed in PBS and incubated with TdT enzyme at 37°C for 1 h in a humidified chamber for incorporation of biotinylated nucleotides at the 3-OH ends of DNA. The slides were incubated in horseradish peroxidase-labeled streptavidin to bind the biotinylated nucleotides followed by detection with stable chromagen DAB. The images on the slides were visualized with an Olympus BX40 light microscope equipped with a computer-controlled digital camera (DP71, Olympus Center Valley, PA, USA). Three slides per group were stained and apoptotic cells were identified by dark brown cytoplasmic staining.

The inflamed skin of mice was collected at the end of the experiment and stored in 10% neutral phosphate buffered formalin. Following fixation, the samples were dehydrated and embedded in paraffin. Microtome sections of about 5 mm thickness were taken from the inflamed skin and stained with hematoxylin as well as eosin. The Olympus BX40 light microscope equipped with computer-controlled digital camera (DP71, Olympus Center Valley, PA) was used to visualize the images on the slides.

Myocardial interstitial fibrosis was identified using Azan Mallory staining. Images were acquired using an Olympus BX40 light microscope (400 x magnification).