Avidin biotin complex

Absolute ethanol was procured from Sigma Chemicals Co., Ltd. (St. Louis, MO, USA). Omeprazole was obtained from a local pharmaceutical company. Avidin-biotin Complex (ABC) and primary antibodies, including rat anti-TNF-α (SC-52B83), rat monoclonal anti-p-NFkB (SC-271908) and rat anti-COX-2 (SC-514489), were acquired from Santa Cruz Biotechnology (Dallas, TX, USA). 3,3-diaminobenzidine peroxidase (DAB), 5,5′-dithiobis (2-nitrobenzoic acid); (DTNB), GSH, and 1-chloro-2,4-dinitrobenzene (CDNB) were acquired from Sigma Aldrich (St. Louis, MO, USA). Secondary antibody was obtained from Abcam (ab-6789, Cambridge, UK), and proteinase K was purchased from MP Bio, USA. H⁺/K⁺-ATPase activity assay screening kit (Catalog No: E-BC-K122-S), Rat p-NFkB ELISA kit (Catalog No: E-EL-RO674), and rat TNF-α ELISA kit (Catalog No: E-EL-R0019) were procured from Elabscience Biotechnology (Wuhan, China). Protein assay kit BCA (Catalog No: 23227, Waltham, MA, USA) was also used. All other reagents utilized were of analytical grade.

To evaluate degree of vascularization in the heart, samples of left ventricle were used. So, 4 μm-thick slices were prepared from the samples. In the next step, these sections underwent processing steps such as deparaffinization, dehydration. To suppress endogenous peroxidase activity, incubation with proteinase K and treatment with hydrogen peroxide 0.3% were applied.
The primary antibody CD31 (Santa Cruz, USA) was used as an indicator of angiogenesis to cover the samples. Later on the sections were incubated at 4°C for 12 hours. Then hatched with usual avidin–biotin complex (ABC; Santa Cruz) giving to the manufacturer’s orders. In the next step, light microscope (Olympus BX 40, Japan) was used for evaluating the strength scoring for CD31 staining (at magnification 40×). For this purpose, 3 to 5 1 mm² areas were selected and staining strength each (at 200×magnification) and amount of positive cells were calculated. As a distinct container of the positive endothelial cell batch of immunoreactivities in interaction alongside with the elected region (1 mm²) was calculated. The granulated tissue was used as a positive control to measure the proportion of immunostaining, and the strength of staining was scored as 0 ( < 10%); 1 (10% to 25%); 2 (25% to 50%); 3 (50% to 75%) or 4 (75% to 100%).^{26 (link)}

The chemicals and reagents used in the current study include UMB, PiCl, histamine, ketamine, dimethylsulphoxide (DMSO), Toluene, dexamethasone, acetone, ethanol, 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (cDNB), phenylenediamine and Tris-HCl were obtained from the Sigma Aldrich (Sigma Aldrich, USA). The avidin-biotin complex (ABC), proteinase-K, 3,3-diaminobenzidine (DAB), xylene, and mounting media were purchased from the Santa Cruz (Santa Cruz, Inc). All the chemical and reagents used in the present study were of analytical grade. The PiCl solution was prepared in acetone and ethanol at 3:1 ratio, respectively.

To investigate angiogenesis in the myocardium, transversal sections of the
ventricles at their midportion were immediately isolated and fixed in 10%
buffered-formalin solution, dehydrated in ascending grades of alcohol and
embedded in paraffin. Then, serial 3 µm-thick sections were cut from them
and floated onto charged glass slides according to standard histological
processing. Tissue pieces were deparaffinised in xylene and dehydrated in a
graded series of ethanol. Slides were incubated sequentially in proteinase K and
0.3% hydrogen peroxide to block endogenous peroxidase activity. Sections were
overlaid by primary antibody CD31 (Santa Cruz, USA) - an angiogenesis marker -
and incubated at +4°C overnight. Afterwards, the sections were washed and
incubated with standard avidin-biotin complex (ABC; Santa Cruz) according to the
protocol. Then the slides were incubated in DAB (Diamino-benzidine, Santa Cruz)
as the chromagen, and counterstained with Mayer's hematoxylin. Finally, the
sections were cleared in xylene, mounted with Entellan and analyzed with a light
microscope.