Horseradish peroxidase conjugated goat anti rabbit igg

Immunohistochemistry was performed on paraffin sections of human quadriceps muscle isolated from patients with OA, as previously described 27. In brief, sections were incubated with rabbit polyclonal antibodies against 11β‐HSD1 (1:50, 10007815, Lot no. 0452916; Cayman, Ann Arbor, Michigan, USA), and then with goat anti‐rabbit biotin as a secondary antibody (1:1000, AB360; The Binding Site, Birmingham, UK). Antigen retrieval was performed with 0.2 × trypsin in 1% (w/v) calcium chloride for 30 min at room temperature and blocking with 10% goat serum. Sections were incubated with horseradish peroxidase‐conjugated goat anti‐rabbit IgG (1:1000; Dako, Glostrup, Denmark) before visualization by incubation with 3,3′‐diaminobenzidine (Vector Laboratories, Burlingame, CA, USA) until development of a brown precipitate (5 min).

To detect caspase-3 positive cells in liver and kidney, immunohistochemistry was performed on 3 μm sections of paraffin embedded liver samples. Sections were deparaffined in a sequence of xylene, alcohol and water. As an antigen retrieval method we used for caspase-3 samples: EDTA (1 mM, pH 8.0) buffer. Next, sections were stained with Caspase-3 primary Antibody (Cell Signaling cat. nr. 9661, 100× diluted in 1 % BSA/PBS) using an indirect immunoperoxidase technique. Endogenous peroxidase was blocked using H₂O₂ 0.3 % in phosphate-buffered saline for 30 min. After thorough washing, sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG as a secondary antibody for 30 min (Dako, Glostrup, Denmark. cat. nr. P0448), followed by rabbit anti-goat IgG as a tertiary antibody for 30 min (Dako, Glostrup, Denmark. cat. nr. P0449). The reaction was developed using DAB as chromogen and H₂O₂ as substrate. Sections were counterstained using Mayer hematoxylin solution (Merck, Darmstadt, Germany). Negative antibody controls were performed. Localization of immunohistochemical staining was assessed by light microscopy. For each tissue section, positive cells per field were counted by a blinded researcher in ten microscopic fields of the tissue at 10× magnification. Results were presented as number of positive cells per field.

Zirconia balls were added to the proteins extracted from the skin of the Nlrp1b KI mice and WT mice at 5 weeks of age (Day 0) before UVB irradiation and at Day 5 after UVB irradiation. Then, the proteins were dissolved in 1 ml sample buffer (NuPAGE LDS sample buffer 250μL, sample reducing agent 100μL, 25×protease inhibiter 40 μL, and water 610 μL) and crushed. After centrifugation at 10,000 rpm for 10 min at 4°C, the supernatant of each sample was subjected to SDS-PAGE. Strips of membrane were incubated with anti-IL-1beta (ab9722; Abcam), anti-pro caspase-1 + p10 + p12 (ab179515; Abcam), or anti-GAPDH (ab9485; Abcam) antibodies. The antibody–antigen complexes were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (Dako, Glostrup, Denmark) at a dilution of 1:1,000, followed by detection with enhanced chemiluminescence western blotting substrate (GE Healthcare BioSciences, Little Chalfont, UK), as described by the manufacturer. n=2. Each experiment was performed three times.

Zirconia balls were added to the proteins extracted from the liver of P0 newborns. Then, the proteins were dissolved in 1 ml sample buffer (NuPAGE LDS sample buffer 250μL, sample reducing agent 100μL, 25×protease inhibiter 40μL, and water 610μL) and crushed. After centrifugation at 10,000 rpm for 10 min at 4°C, the supernatant of each sample was subjected to SDS-PAGE. Strips of membrane were incubated with anti-p-JAK1 (Tyr1021), anti-JAK1 (ab125051; Abcam), anti-p-STAT1 (Tyr701), anti-STAT1 (ab99415; Abcam), anti-p-STAT3 (Tyr705), anti-STAT3 (SAB4300708, Sigma Aldrich), anti-p-STAT5 (Tyr694), anti-STAT5 (ab16276; Abcam), anti-p-STAT6 (Tyr641), anti-STAT6 (ab32520; Abcam) or anti-GAPDH (ab9485; Abcam) antibodies. The antibody–antigen complexes were detected with horseradish peroxidase–conjugated goat anti-rabbit IgG (Dako, Glostrup, Denmark) at a dilution of 1:1,000, followed by detection with enhanced chemiluminescence Western blotting substrate (GE Healthcare BioSciences, Little Chalfont, UK), as described by the manufacturer. n=3. Each experiment was performed three times. For HEK293 cell lysates, additional anti-HaloTag monoclonal antibody (G9211; Promega Corporation, WI) and anti-JAK1 rabbit monoclonal antibody (#3344; Cell Signaling Technology, MA) were used as the primary antibodies.