Rtu vectastain kit

IHC staining was performed according to the previous procedures (31 (link)). Briefly, 4-μm-thick sections of tumor tissues were cut from FFPE blocks and mounted on slides. All slides were dried for 2 h at 62 °C. Sections were subsequently deparaffinized in xylene and rehydrated in graded alcohol. Antigen retrieval was achieved by heating the slides in a target retrieval solution (pH 6.0; 0.01 mol/L citrate buffer) for 15 min, then cooling them for 90 min at room temperature. After the endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide in methanol for 10 min, nonspecific binding was blocked by incubating the slides with 5% bovine serum albumin in phosphate-buffered saline (PBS) for 30 min. After the specimens were washed three times with PBS, they were reacted overnight at 4 °C with primary mouse anti-human monoclonal CD8, CD4, and CD3 antibody (1:200 solution; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with a biotin-conjugated secondary antibody for 30 min at room temperature, sections were further treated with an avidin-biotin-peroxidase complex system (RTU VECTASTAIN kit, VECTOR LABORATORIES, Burlingame, CA, USA). Finally, the signal was developed with 3,3´-diaminobenzidine tetrahydrochloride (1:50 solution; DAB Substrate kit, Abcam, Cambridge, MA, USA). All sections were then counterstained with hematoxylin and mounted.

Hearts from the euthanized mice were fixed in 4% formaldehyde (pH 7.3) for 12 h, dehydrated in alcohol, clarified in xylene, and embedded in paraffin to be sectioned at 5 μm. Protein levels were evaluated in cardiac tissues using the immunoperoxidase technique with anti-p-MYPT1 (T696) (Cell Signaling, USA #5163), anti-ROCK1 (Cell Signaling, USA #4035), anti-ROCK2 (Cell Signaling, USA #9029) antibodies. Staining was performed using a peroxidase and diaminobenzidine kit with a chromophore according to the manufacturer’s instructions (RTU-Vectastain kit; Vector Laboratories, USA). The heart tissue was additionally stained with hematoxylin. The images were obtained with a Nikon Eclipse 400 epifluorescence microscope (Nikon, Japan) and analyzed with ImageJ software (ImageJ 1.47v).

Mouse organs were fixed in 10% formalin for 24 hours. Tissues were processed using a Leica ASP300S Tissue Processor, paraffin embedded, and cut into 4-μm sections. H&E staining was performed using hematoxylin Solution, Gill No. 3 (Sigma-Aldrich) and Eosin Y (Thermo Fisher Scientific). Immunohistochemistry (IHC) was performed manually following deparaffinization and rehydration. Antigen retrieval was done in citrate buffer pH 6.0 (Vector Laboratories) using a Decloaking Chamber (Biocare Medical). Endogenous peroxidase activity and biotin were blocked using H₂O₂ and the Avidin/Biotin Blocking Kit (Vector Laboratories), respectively. Following incubation with primary antibodies, the R.T.U. Vectastain Kit (Vector Laboratories) and DAB in chromogen solution (Dako) were used to develop the signal. The sections were then counterstained with hematoxylin (Dako). For quantification of T cells, ImageJ was used to count cells in eight ×20 microscopic fields per mouse.