Goat anti rabbit igg h l mouse human ads hrp

Formalin fixed, paraffin embedded tissue blocks were prepared from mouse skin 2 days post-infection as follows: Tissue samples were fixed in 4% paraformaldehyde overnight, embedded in paraffin, and prepared for histological analysis. For immunohistochemistry, 2-micron sections were immune-labeled using routine immunoperoxidase methods. Briefly, the paraffin was removed from sections with xylenes, after which they were washed in ethanol and then rehydrated in PBS. Samples were blocked 30 min in 3% H₂O₂ solution (Sigma-Aldrich), blocked with 10% goat serum/TRIS-buffered saline for 15 min, incubated for 2 h at room temperature with the primary antibodies, incubated 1 h with species-specific secondary antibodies and lastly developed in 3,3′-diaminobenzidine tetra hydrochloride as a chromogen. Primary antibodies used: anti-Myeloperoxidase (rabbit 1:50 ab9535, Abcam) and anti-histone H3 (citrulline R2 + R8 + R17) (rabbit 1:50 ab5103). Secondary antibody used: Goat-anti-rabbit IgG (H&L) mouse/human ads-HRP (1:200, Southern Biotech).

Approximately 200 mg of liver tissue was added to 2 mL of RIPA lysate and 20 μL of protease inhibitor. The ultrasonic homogenizer was used to homogenize the sample in an ice bath until no granules were detected. After centrifugation, the middle layer of the liquid was collected (the upper layer was oil, and the lower layer had a little precipitation), and centrifugation was repeated one time. For the BCA method and protein hyperthermia degeneration, the cells were transferred to a PVDF membrane and 5% skim milk powder at room temperature for 1.5 h. The membrane was immersed in the TLR4 antibody 1:500, NLRP3 antibody 1:500, ASC antibody 1:300, Caspase-1 antibody 1:500, or NF-κB p65 antibody 1:1000 at 4°C overnight. After washing the membrane, goat anti-rabbit IgG (H+L), mouse/human ads-HRP (1:20,000; Southern Biotech, 4050-05), or rabbit anti-mouse IgG (H+L)-HRP (1:10000; Southern Biotech, 6170-05) was incubated with the membrane for 1.5 h at room temperature. After washing the film, ECL luminescence solution was added, and the film was developed. The semiquantitative analysis of the bands was performed using software. The OD value of the target protein was divided by the OD value of the β-actin band as the final result.