Anti asc ab

AM were fixed with 4% paraformaldehyde for 20 min. After washing with PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature, followed by blocking with 3% bovine serum albumin in PBST (PBS with 0.1% Tween-20) for 2 h at room temperature to reduce non-specific staining. The cells were then incubated with rabbit polyclonal anti-ASC Ab (Santa Cruz Biotechnology) at 4 °C overnight. After washing with PBS, the cells were incubated with Alexa Fluor 555-conjugated donkey anti-rabbit IgG (Abcam) for 1 h at room temperature. Hoechst 33258 (Sigma) was used to stain nuclei. The cells were then washed with PBS, followed by confocal microscopy. For visualize AM pyroptosis, the cells were incubated with Alexa Fluor 488-labeled caspase-1 FLICA at 37 °C for 1 h. After fixation with 4% paraformaldehyde, cells were stained with TMR red-labeled In-Situ Cell Death Detection reagent (Roche Applied Science, Indianapolis, IN) following the manufacturer’s instructions. The cells were then analyzed by confocal microscopy.

Mouse lung tissue or MLVEC were homogenized or lysed (1 × 10⁶ cells per ml) in lysis buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 mM NaF, 1 mM Na3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 20 mM PMSF). The supernatants were quantified, and 600 μg total protein for each sample was then immunoprecipitated with anti-ASC Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-TXNIP Ab (MBL International, Ottawa, IL, USA). The immunoprecipitated proteins were separated on a 10% SDS-PAGE gel and then electroblotted onto polyvinylidene difluoride membrane and blocked for 1 h at room temperature with Odyssey Blocking Buffer (LI-COR Biotechnology, Lincoln, NE, USA). Nlrp3 was detected by probing the membranes with anti-Nlrp3 Ab (Santa Cruz Biotechnologies) at 1 : 500 dilution and detected with fluorescent secondary antibody (LI-COR Biotechnology) following the manufacturer's instructions. Blots were then stripped and reprobed with anti-ASC Ab or anti-TXNIP Ab and again detected with fluorescent secondary antibody (LI-COR Biotechnology). Caspase-1 cleavage in the lung tissue or MLVEC was measured by detecting its p10 fragment by western blot using rabbit polyclonal anti-mouse caspase-1 p10 (Santa Cruz Biotechnologies). TXNIP protein in MLVEC was detected by western blot using anti-TXNIP Ab.

Cells were prepared as described and infected with PmCQ2 for 3 or 4 h. After infection, cells were fixed with 4% paraformaldehyde (Sango Biotech, Shanghai, China) for 30 min at room temperature (RT) and then blocked with 5% Bovine Serum Albumin (BSA) for 1 h at RT. After washing steps, primary antibodies (anti-ASC Ab, Santa Cruz, CA; anti-NLRP3 Ab, Wanlei Life Sciences, Shenyang, China) were added and incubated overnight at 4°C. Next, Goat anti-rabbit IgG (H&L) Alexa fluor 594 (Abcam, United Kingdom) was added after washing with PBS at RT for 1 h. Subsequently, DAPI (Beyotime Biotechnology, Shanghai, China) was added and incubated in the dark for 5 min. Finally, anti-fluorescence attenuation mounting tablets (Solarbio, Beijing, China) were used and the results were observed an inverted fluorescence microscope (Olympus, Tokyo, Japan).
To show the extent of speck formation more intuitively, the percentage of cells that contained a speck was determined. Cells from five different fields (average of 100 cells/field) were counted based on DAPI-stained nuclei for each of different experiments. Images were analyzed using ImageJ. The data is expressed as the percentage of cells with specks per number of cells per field.