Anti asc

Mice were deeply anesthetized with isoflurane and perfused intracardially with PBS followed by 4% paraformaldehyde. Brains were removed, post-fixed overnight in 4% paraformaldehyde followed by an additional overnight incubation in 30% sucrose solution at 4 °C, and then stored at −80 °C. Frozen brains were coronally sectioned in a cryostat microtome at 35 μm. Slices were subsequently washed with PBS and blocked using 10% normal donkey serum prepared in PBS containing 0.1% Triton X-100 for 1 h at room temperature. Next, slices were incubated with the appropriate primary antibody (anti-NeuN, 1:1000, Synaptic Systems, 266004; anti-IBA1, 1:1000, FUJIFILM Cellular Dynamics, 019-19471; anti-GFAP, 1:1000, Cell Signaling Technology, 12389; anti-ASC, 1:200, AdipoGen, AG-25B-0006-C100; anti-CASP-1, 1:1000, Santa Cruz, sc-56036; anti-mCherry, 1:1000, Millipore Sigma, AB356481) in 10% NGS PBS overnight at 4 °C on a shaker. Then slices were washed three times (10 min each) with PBS and incubated with the Alexa Fluor 488 and 594 conjugated secondary antibodies (1:1000, Invitrogen, A-21206, A-11042, A-11076) in 10% NGS PBS for 2 h at room temperature. Following three washes (15 min each) with PBS, slices were mounted on superfrost plus slides and covered with Vectashield mounting medium (Vector Laboratories, Burlingame, USA) containing DAPI. Slides were stored at 4 °C until imaging.

Anti-β-actin (1:10,000, BH10D10) was bought from Cell Signaling Technology (Danvers, MA, United States); Anti-NLRP3 (1:1,000. Cryo-2) and Anti-ASC (1:1,000, AL177) were purchased from Adipogen (San Diego, CA, United States); Anti-Caspase-1 (1:1,000, ab179515) and Anti-NEK7 (1:10,000 ab133514) were bought from Abcam (Cambridge, CB2 0AX, United Kingdom); Anti-IL-1β (1:000 AF-401-NA; RRID: AB_416684) was obtained from RD systems (Tustin, CA, United States); the DyLight 488-labeled secondary antibody (1:50, A120-100D2) was purchased from InvivoGen (San Diego, CA, United States); and FITC anti-mouse/human CD11b (101216, 1:500 for flow cytometry) and APC anti-mouse Ly-6G (127614, 1:500 for flow cytometry) were from BioLegend.

The kidney tissues were homogenized and the supernatant was collected after centrifugation at 12,000 ×g at 4°C for 20 min [23 (link)]. We separated the lysates on 10% polyacrylamide gels before immunoblotting using anti-Nlrp3 (AdipoGen company, San Diego, CA), anti-ASC (AdipoGen company, San Diego, CA), anti-CHOP (Cell Signaling Technology, USA), anti-caspase-12 (Cell Signaling Technology, USA), anti-IL-1β (Affinity Biosciences, USA), and anti-IL-18 (Affinity Biosciences, USA) antibodies at a dilution of 1 : 500. The expression levels of CHOP, caspase-12, ASC, Nlrp3, IL-1β, and IL-18 were analyzed using an ECL advance system (Amersham, Little Chalfont, UK). The relative protein expression levels were determined by normalization to β-actin. Serum IL-1β and IL-18 were measured with ELISA kits (RayBiotech, Norcross, GA) according to the manufacturer's instruction.

BMDCs (2 × 10⁶) were treated with or without blocking antibody for 60 min before the addition of live or heat-killed H. capsulatum. For inflammasome analysis, cells were cultured in medium containing 0.1% heat-inactivated FBS and for signaling molecule analysis, in medium containing 10% FBS. Cells were detached from the wells and lysed with PhosphoSafe lysis buffer (MERCK) at different time points. Harvested cell-free supernatants were concentrated by 10-fold with Vivaspin 500 (GE Healthcare). Cell lysates were subjected to electrophoresis at 10% (for cell lysates) or 12.5% (for supernatants) SDS polyacrylamide gel and transferred to a 0.45 (for cell lysates) or 0.22 (for supernatants) μm PVDF membrane. The membrane was blocked with 5% non-fat milk and left in buffer containing anti-IL-1β p17 (R&D system), anti-Caspase-1 p20 (Adipogen), anti-NLRP3 (Adipogen), anti-ASC (Adipogen), anti-p-Syk (Abcam), anti-p-ERK (Cell Signaling), anti-p-JNK (Cell Signaling), anti-p-p38 (Cell Signaling) or anti-β-actin (GeneTex) antibody at 4°C overnight. The membrane was washed with TBST before addition of HRP-conjugated anti-goat IgG (1:3000), anti-rabbit (1:20,000) or anti-rat IgG (1:20,000). ECL reagent (PerkinElmer Life Science, Merck Millipore and GE Healthcare) was used for detection.

Lung tissue was lysed in Pierce™ IP Lysis Buffer (ThermoFisher Scientific). The lysates were centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected and the protein concentration in the supernatant was determined by BCA Protein Assay Kit (Bio-Rad Laboratories). For the immunoblot, 40 µg of the total protein was resolved in SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat milk in PBS containing 0.1% Tween-20 for 30 min at room temperature. The membrane was incubated with primary antibodies overnight at 4 °C followed by incubation with secondary antibodies. The membrane was then developed using ECL Western Blot Substrate (ThermoFisher Scientific). The following primary antibodies were used: anti-NLRP3 (1:1000, AG-20B-0014-C100), anti-Caspase-1(p20) (1:1000, AG-20B-0042-C100), anti-Asc (1:500, AG-25B-0006-C100) from Adipogen Life Sciences, Anti-p65 (8242, 1:1000), anti-P-p65-Ser536 (3033, 1:500) from Cell Signaling Technology Inc., and anti-β-actin (1:5000, A5441, Sigma-Aldrich). For the immunoblot analysis of BALF samples, equal volumes of BALF from all groups were loaded. The following primary antibodies were used: anti-Histone H2B (1:500, ab52484, Abcam), anti-CitH3 (1:500, ab5103, Abcam),