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5 protocols using mouse anti α sma

1

Inflammasome Activation Assay

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Nigericin, ATP, poly(dA:dT), poly(I:C), phorbol-12-myristate-13-acetate (PMA), dimethyl sulfoxide (DMSO) and ultrapure LPS were obtained from Sigma-Aldrich (Munich, Germany). Silicon dioxide (SiO2) and Pam3CSK4 were obtained from InvivoGen (Toulouse, France). MCC950, carnosol, and geldanamycin (GA) were obtained from TargetMol (Boston, MA, USA). MitoTracker and MitoSOX were manufactured by Invitrogen (Carlsbad, CA, USA). Salmonella was a gift from Dr. Tao Li of the National Center of Biomedical Analysis. Anti-mouse Caspase-1 (1:1000, AG-20B-0042) was from Adipogen (San Diego, USA). Anti-human cleaved IL-1β (1:2000, 12242), Anti-mouse α-SMA (1:1000, 19245s), anti-human Caspase-1(1:2000, 4199S), anti-mouse IL-1β (1:1000, 12507) and anti-NLRP3 (1:2000, 15101S) were from Cell Signaling Technology (Boston, USA). Anti-ASC (1:1000, sc-22514-R) was from Santa Cruz Biotechnology (Dallas, USA). Anti-DDDK tag (1:3000, 20543–1-AP), Anti-HSP90 (1:3000, 13171-1-AP) and Anti-GAPDH (1:2000, 60004-1-1G) were from Proteintech Group (Chicago, USA).
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Investigating Murine IL-17A Signaling Pathways

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The mouse IL-17A protein was purchased from Peprotech (Rocky Hill, NJ). The neutralizing mouse IL-17A mAb was obtained from R&D Systems (Minneapolis, MN). Anti-mouse α-SMA, IL-17R, RORγt, p-STAT3, STAT3, LC3 II/I, Beclin-1, LAMP-1, Vps34, CD11b, CD206, and β-actin Abs were purchased from Cell Signaling Technology (Danvers, MA). Anti-mouse p62 Ab and thioacetamide (TAA) were purchased from Sigma (St. Louis, MO). Alexa Fluor 488 and 647 Abs were obtained from Invitrogen (San Diego, CA). The assay kits for Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The ELISA kits for IL-17A, TGF-β1, IL-6, IL-23, IL-13, IL-10 and IFN-γ were purchased from eBioscience (San Diego, CA). Other materials were purchased from commercial sources.
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Immunohistochemical Analysis of Tissue Markers

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Four micrometer-thick slides were deparaffinized with xylene before rehydration using an ethanol gradient. Quenching of the endogenous peroxidase activity was achieved by incubating samples with endogenous peroxidase blocker. After blocking with 3% BSA, sections were incubated with mouse anti-α-SMA (Cell Signaling Technology, Boston, MA, USA), mouse anti-TGF-β (R&D, Minneapolis, MN, USA), rabbit anti-VEGFA (Abcam, Cambridge, UK), and mouse anti-FGF basic/FGF2 (Novus, Littleton, CO, USA) overnight at 4 °C. Horseradish peroxidase-conjugated goat anti-mouse/rabbit lgG was used as the secondary antibody. The DAB Substrate System (DAKO) was used to reveal the immunohistochemistry staining.
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Immunofluorescence Analysis of Lin28a, α-SMA, and C/EBP in Paraffin-Embedded Tissue

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Paraffin-embedded sections (4 μm) were de-paraffinized with xylene and rehydrated with gradient concentrations of alcohol. The sections were then placed in 0.01 mol/L citrate buffer for high-pressure repair. Endogenous peroxidase activity was blocked using H 2 O 2 . Sections were incubated overnight with rabbit anti-Lin28a (1:200, Proteintech, Wuhan, China), mouse anti-α-SMA (1:200, Cell Signalling Technology, Danvers, MA, USA), mouse anti-C/EBP (1:200, Proteintech), and rabbit anti-α-SMA (1:200, Cell Signalling Technology) at 4°C. After washing with phosphate buffered saline (PBS), the sections were incubated with goat anti-mouse IgG H&L (FITC) (1:500, Abcam, Cambridge, UK) or goat anti-rabbit IgG H&L (Alexa Fluor ® 647) (1:500, Abcam) for 1 h. Finally, the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Solarbio) for 5 min.
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5

Immunofluorescence Analysis of Pulmonary Fibrosis

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Cryosections of lung tissues from IPF patients and mice with the onset of BLM-induced pulmonary fibrosis were used for immunofluorescence staining. The primary antibodies used for staining were as follows: mouse anti-ACP5 (Abnova, Taipei, China, 1: 100), rabbit anti-ACP5 (Proteintech, Wuhan, China, 1:100), rabbit anti-FSP1 (Proteintech, Wuhan, China, 1:100), mouse anti-α-SMA (Cell Signaling Technology, MA, USA, 1:100), rabbit anti-β-catenin (Cell Signaling Technology, MA, USA, 1:100), and rabbit antiphospho-Smad3 (Cell Signaling Technology, MA, USA, 1:100). Alexa 488- or 594-conjugated anti-mouse or rabbit (Abbkine, CA, USA, 1:400) were used as fluorescent secondary antibodies, and nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI). Images were obtained with a fluorescence microscope (Olympus, Shinjuku, Japan).
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