Abc kit

The kidneys were fixed with 10% formalin and embedded in paraffin. Kidney sections (4-μm thickness) were incubated in 3% H₂O₂ for 15 minutes at room temperature to block endogenous peroxidase activity. After boiling in antigen retrieval solution (1 mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH = 8.0) for 15 minutes at high power in a microwave oven, the sections were incubated overnight at 4 °C with a rabbit anti-NKCC2 antibody (Stressmarq Biosciences Inc., Canada) or a rabbit anti-ENaCα antibody (Stressmarq Biosciences Inc., Canada). After washing with PBS, the secondary antibody was applied, and the signal was visualized using an ABC kit (Santa Cruz Biotechnology).

The kidneys were fixed in 10% formalin and embedded in paraffin. Kidney sections (4-μm thickness) were deparaffinized, hydrated in ethyl alcohol and washed in tap water. Endogenous peroxidase activity was blocked in 3% H₂O₂ for 15 min. Antigen retrieval was carried out in boiling antigen retrieval solution (1mmol/L Tris-HCl, 0.1mmol/L EDTA, pH = 8.0) for 15minutes. Then the sections were incubated overnight at 4°C with rabbit anti-NKCC2 antibody (Catalog No.: SPC-401, Stressmarq Biosciences Inc., Canada) and at room temperature for 30 min with secondary antibody. Staining was visualized using an ABC kit (Santa Cruz Biotechnology).

Interstitial cells of Cajal (ICC) are cells distributed in the smooth muscle layer of the intestine that assists in intestinal peristalsis [13 (link)]. To observe these cells, paraffin-embedded intestinal tissues were cut into 4 μm sections after staining with H & E. The sections were then deparaffinized using xylene and hydrolyzed for 5 min with different concentrations of ethanol (100%, 90%, 80%, and 70%). Slide glass containing the tissue was heated to 98°C for 20 min for antigen retrieval, followed by preantibody blocking to prevent staining of cells other than ICC. Primary antibody was diluted 1 : 200 using AB c-kit (Santa Cruz; SC-168, Dallas, TX, USA) and reacted at 4°C for 1 day. After the reaction, antibody enhancer was added to the tissue and incubated for 10 min. After 5 min of treatment with chromogen (DAB Substrate 1 ml + DAB chromogen 1-2 drops), the sections were washed in running water for 1-2 min, reacted with hematoxylin, and then washed with PBS and running water, and eventually they were mounted. Stained ICCs were observed under an optical microscope (ZEISS, Axiovert S100, Jena, Germany), and the number of pixels with RGB values was determined using MATLAB.

Immunohistochemical staining was achieved according to our previously published report [18 (link)]. Briefly, after the deparaffinization protocol described above, slides were treated with proteinase K to clear formaldehyde remains from the antigen epitope and then washed. A diluted H₂O₂ solution (3% in methanol) was employed for quenching peroxidase reactivity. After washing, slides were treated with a normal serum-like (NGS). Slides were then incubated for a whole night with primary antibodies, as demonstrated in material sections with a dilution factor of 1:100. The next morning, slides were consecutively treated with a biotin-tagged 2° antibody (dilution 1:50) and with an ABC kit (Santa Cruz Biotechnology) and then stained in DAB solution. This step was followed by gradient ethanolic dehydration and fixation in absolute xylene (reverse deparaffinization protocol) and mounting with a glass coverslip. By using an Olympus light microscope at 40× magnification scale, slides were analyzed for hyperactivated p-JNK, TNF-α, caspase-3, and COX2 using an ImageJ program [19 (link)].

We employed a previously described procedure with slight modifications for immunohistochemical analysis [56 (link)]. After completion of the deparaffinization step, slides were processed by an enzymatic method for antigen retrieval and then washed with PBS consecutively three times for 5 min. Slides were immersed in 3% H₂O₂ to quench endogenous peroxidase activity followed by washing with PBS. Normal goat serum (5%) was applied as a blocking serum, and slides were incubated for 2 h. Next, the slides were incubated overnight with primary antibodies Bcl2, p-JNK, TNF-α, Nrf2, HO-1, and COX-2. The next morning, slides were washed with PBS and incubated for 90 min with the secondary antibody, then incubated with an ABC kit (Santa Cruz) in a humidified box for 60 min. Slides were then washed with PBS solution and stained with DAB, followed by dehydration with ethanol (70%, 80%, 90%, and 100%). After dehydration, slides were fixed with xylene and then cover slipped with mounting media. Images were obtained using a light microscope and saved in TIFF format for further quantification by the ImageJ software.