Prs3459

For immunofluorescence staining, the cells were fixed with 4% paraformaldehyde for 15 min in PBS, permeabilized with 0.2% Triton X-100 in PBS, blocked with blocking buffer and then stained with primary antibodies against α-tubulin (ab52866, Abcam), α-actinin (A7811, Sigma-Aldrich), or cleaved caspase-1 p20 (PRS3459, Sigma-Aldrich) followed by Alexa Fluor 488/546/633 secondary antibodies. ASC was detected with an Alexa Fluor 488 Conjugated antibody (17507, Cell Signaling). Rhodamine phalloidin dye (R415, Invitrogen, Carlsbad, CA, USA) was incubated with the cells for 20 min at 37 °C for F-actin. The stained cells were visualized and analyzed using a laser scanning confocal microscope with a 63×/1.4NA oil immersion objective lens and excitation wavelengths of 488, 543, and 633 nm. All images were acquired using ZEN 2012 software.

The ipsilateral brain tissues were removed 24 h after ICH and lysed in RIPA lysis buffer (Biocolors, 11,814,389,001, Shanghai, China). Protein extracts were electrophoretically resolved with SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Roche, GVWP02500, Basel, Switzerland). After blockade with 5% non-fat milk, the blots were then incubated with primary antibody against NLRP1 (1:2000, ABF22, Sigma, Missouri, USA), ASC (1:2000, SAB4501315, Sigma), pro-caspase-1 (1:2000, PRS3459, Sigma), caspase-1 (1:2000, AB1871, Sigma), or β-actin (1:2000, A1978, Sigma) overnight at 4 °C, followed by peroxidase-conjugated secondary antibody (Cell Signaling Technology, MA, USA). The blots were visualized using an electrochemiluminescence detection kit (Amersham, Little Chalfont, UK).

After ISO treatment for 1 h, the mice were anesthetized with 3% isoflurane in oxygen, and the pedal pinch reflexes were completely inhibited before euthanasia. The heart tissues were then isolated and fixed with optimal cutting temperature compound (4583, SAKURA, Torrance, CA, USA). Frozen sections of the mouse hearts (6-μm thick) were obtained using a cryostat (Leica, Wetzlar, Germany), placed on poly-L-lysine-coated glass slides, soaked with acetone, permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 10 min, blocked with 5% BSA and then stained with primary antibodies against vimentin (1:100 dilution, ab8979, Abcam, Cambridge, MA, USA), cleaved caspase-1 p20 (1:200 dilution, PRS3459, Sigma-Aldrich), and the nuclei dye Hoechst 33342. The frozen sections were visualized using a laser scanning confocal microscope (LMS 780, Carl Zeiss, Inc., Thornwood, NY, USA) with a 63×/1.4NA oil immersion objective lens and an excitation wavelength of 405/488/543 nm. All images were acquired with ZEN 2012 software. The immunofluorescence staining and image capturing were carried out without knowledge of treatments.