Dmi3000b inverted fluorescence microscope

The fluorogenic substrate 2′,7′-dichlorofluorescein diacetate is a cell-permeable dye, which, after entering into a cell, can be hydrolyzed into non-fluorescent 2′,7′-dichlorofluorescein by intracellular esterases and then oxidized to fluorescent 2′,7′- dichlorofluorescein by ROS within the cytosol; the resulting change in fluorescence intensity can be used to measure intracellular generation of ROS. To assay for ROS, H441 cells were cultured in 24-well plates, as described above. After exposure to 200 μM formaldehyde for 24 h, the cells were washed twice with PBS and then incubated in medium containing 10 μM 2′,7′-dichlorofluorescein diacetate for 30 min at 37 °C in the dark. Immediately after incubation, the cells were washed three times with serum-free medium to remove extracellular 2′,7′-dichlorofluorescein diacetate, and fluorescence intensity was monitored using a Leica DMI 3000B inverted fluorescence microscope.

The bEnd.3 cells were plated on the coverslips in a 3.5 cm dish. Following the OGD-re treatment, as previously described, the coverslips were washed with phosphate buffer saline (PBS) then fixed in 4% paraformaldehyde for 30 min at room temperature (RT). The coverslips were permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min at 4 °C. Positive control for the TUNEL assay was incubated in 50 μL RQ1 RNase-free DNase (50 U/mL) for 10 min at RT. The positive control and treated coverslips were then washed in PBS and incubated with a 50-μL TUNEL reaction mixture, which contained a fluorescein reconjugated nucleotide mixture and the enzyme terminal deoxynucleotidyl transferase (TdT), prepared by following the manufacturer’s guidelines, for 1 h at 37 °C in the dark. The coverslip, which was not exposed to the TdT enzyme, would be a negative control. After washing with PBS, the coverslips were covered on microslides with a DAPI-Vectashield mounting medium in order to be examined with a DMI3000B inverted fluorescence microscope (Leica).

Cells were seeded on coverslips (150 000 cells per coverslip) and treated as described above with the addition of 25 μM Z-VAD-fmk (Bachem) to prevent detachment of pyroptotic cells and therefore loss of cells with ASC specks. Coverslips were fixed with 4% paraformaldehyde (PFA) (15 min, 37 °C, Alfa Aesar) and washed with PBS. Nuclei were stained with Hoechst 33342 (Life Technologies) for 10 min and the slides mounted using Vectashield (Vector Laboratories). For quantifications of ASC aggregates (specks or filaments), ten random regions of interest were imaged at × 20 magnification (Leica DMI3000B inverted fluorescence microscope, HCX PL FLUOTAR objective, Leica DFC3000G camera and LAS AF Version 3 software) and the number of ASC aggregates and cells were counted. For representative images, the slides were imaged at × 63 magnification (Leica point scanning confocal ‘SP8', HC PL APO CS2 × 63 objective, Leica AF software version 3). For measurements of speck sizes, random regions of interest were images at × 63 magnification (Leica point scanning confocal ‘SP8') and the largest diameters of individual specks were measured using Fiji63 (link).

ZF4 cells were seeded on coverslips at a density of 2 × 10⁵ cells per well in 24-well plates and cultured overnight. After infection with indicated bacterial strains, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with 10% FBS in PBS for 20 min. Cells were then incubated with anti-caspy2 antibodies (1:1000; custom-made; Genscript), followed by goat antirabbit IgG (H + L) secondary antibody, oregon green 488 (O-11038, Thermo Fisher Scientific) and 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (C1005, Beyotime Biotechnology). Images were acquired using a Leica DMI3000B inverted fluorescence microscope.
For caspy2 oligomer cross-linking, ZF4 cells were plated on 12-well plates and stimulated as indicated. Cells were lysed with PBS buffer containing 0.5% Triton X-100, and the lysates were centrifuged at 6797×g for 15 min at 4 °C. Supernatants were transferred to new tubes (Soluble fractions). The Triton X-100-insoluble pellets were washed with PBS twice and then suspended in 200 μl PBS. The pellets were then cross-linked at room temperature for 30 min by adding 4 mM dextran sulfate sodium. The cross-linked pellets were centrifuged at 6797×g for 15 min and dissolved directly in SDS sample buffer.

A histopathological examination of liver tissues was performed. Tissues fixed in 10% formalin were immersed in paraffin blocks, cut into 5 mm thin sections, mounted on glass slides, and stained with hematoxylin and eosin (HE). Tissue sections on slides were observed and photographed using a light microscope (DMI3000B Inverted Fluorescence Microscope: Leica, Germany).

Cells cultured in 24-well plates were fixed with 4% paraformaldehyde for 20 min at room temperature and then permeabilized using 0.5% Triton X-100 for 20 min. The fixed cells were then blocked in 5% BSA for 2 h and incubated overnight at 4 °C in a suitably diluted primary antibody against p-Smad1/5/8 (1:500, Cell Signaling). After washing with PBS, the cells were incubated with Alexa Fluor 647-labelled goat anti-rabbit IgG (H+L) antibody (1:500 dilution; Beyotime, China) at room temperature in the dark for 1 h. The cells were then incubated with DAPI for 10 min and sealed with sealing liquid containing an anti-fluorescence quencher, and images were captured on a Leica DMI 3000B inverted fluorescence microscope.