Horseradish peroxidase conjugated goat anti rabbit igg

Total protein was extracted from the LAAW and RAAW, and the protein concentration was determined by a BCA Protein Assay Kit (AS1086, ASPEN). 10% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed and transferred to a polyvinylidene difluoride membrane, which was incubated with the primary antibodies SK4 (bs-6675R-HRP, Bioss, Inc., China; 1:1000) and p-p38 (Absin#4511, Shanghai, China; 1:1000) overnight at 4 °C. Then, it was incubated with the secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG (Aspen, AS1107), at 37 °C for 45 min after washing with Tris-buffered saline with Tween. GAPDH was used as a control to normalize the signal intensities. An AlphaEase FC software system was used to analyze the optical density value.

All samples for histology were fixed in 4% paraformaldehyde fixative until embedded in paraffin. Four-micrometer sections were cut from paraffin blocks of the left atrial anterior wall (LAAW) and right atrial anterior wall (RAAW). The primary antibody SK4 (bs-6675R-HRP, Bioss, Inc., China, 1:200) and c-Fos (ab209794, Abcam, Inc., UK; 1:100) were added to the sections overnight at 4 °C. Then, the sections were incubated with secondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG (Aspen, AS1107; 1:200) and stained with diaminobenzidine (DAB) solution. The immunological reaction was visualized by DAB staining. Three sections per animal were prepared and four visual fields were randomly selected in every section. The mean density of these slides was determined using computer-assisted IPP 6.0 software. The mean optical density of SK4 = Sum of optical density values/positive cell area. The c-Fos-positive nuclear cardiomyocytes were counted under an optical microscope.