Goat anti rabbit igg h l hrp ab205718

Cells were washed with phosphate‐buffered saline and lysed with protease inhibitor‐containing RIAP lysis buffer (Thermo Science, Rockford, IL, USA). Supernatant was collected after centrifugation at a high speed. Protein quantification was performed by bicinchoninic acid method, and supernatant was heated in a water bath kettle for protein denaturation. Proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto a nitrocellulose (NC) membrane (Millipore, MA, USA) before 30 minutes of blocking with skim milk powder at room temperature. After the membrane was washed with Tris‐buffered saline, Tween 20 (TBST), primary antibodies anti‐TNNT1 antibody (ab83907) (dilution 1:1000; Abcam, Cambridge, UK) and anti‐GAPDH antibody (ab181602) (dilution 1:1000; Abcam) were added for culture at 4°C overnight. Then the membrane was rinsed with TBST solution and then incubated together with secondary antibody Goat Anti‐Rabbit IgG H&L (HRP) (ab205718) (dilution 1:2000; Abcam) at room temperature for 1 hour. Subsequently, color rendering was performed using an enhanced chemiluminescence kit (Pierce, Waltham, MA, USA).

The expression of p65 and S100A9 in mouse skin tissues was detected by immunohistochemistry following the standard protocol. Sections were rehydrated and incubated with primary antibodies against NF-κB/p65 (ab16502, Abcam, UK) and S100A9 (ab92507, Abcam, UK) overnight at 4 ℃. The sections were then incubated with goat anti-rabbit secondary antibody [Goat AntiRabbit IgG H&L (HRP), ab205718, Abcam, UK] for 1 hour at room temperature. Subsequently, sections were washed and counterstained with DAB/hematoxylin (ZSGB-BIO Technology, Beijing, China). Images were captured using NIS-Elements AR (Nikon, Japan) and analyzed by Image J software with IHC Profiler Plugin.