Trekavidin hrp label kit

From tumors of each treatment group, 4-μm-thin tissue sections were cut using a microtome and transferred to gelatin-coated slides [36 (link)]. Tissue sections were incubated with primary antibodies anti-FADD (ab24533), anti-apoptotic protease activating factor 1 (APAF-1) (ab2001), and BCL-2 (ab32124; Abcam, Burlingame, CA, USA) at 4 °C overnight. Slices were washed with phosphate-buffered saline (PBS) and incubated with a streptavidin/Haptoglobin Related Protein (HRP)-conjugated secondary antibody (Biocare Medical, Concord, CA, USA). Immunoreactivity to the various proteins was visualized with a colorimetric-based detection kit following the protocol provided by the manufacturer (TrekAvidin-HRP Label + Kit from Biocare Medical, Pacheco, USA). Light microscopy (Nikon Eclipse 2000 equipped with Nikon DS-Fi2; Nikon Corporation, Tokyo, Japan) with a high-power objective (40×) was used to acquire digital images. The intensity of cell immunostaining was scored as follows: 1 = absence of positive cells, 2 = small number of positive cells or isolated cells, 3 = moderate number of positive cells, and 4 = large number of positive cells. Labelling intensity was evaluated by two previously trained examiners in a double-blind fashion.

Ethanol was purchased from LIBBs Farmacêutica Ltda, São Paulo, Brazil. Carvedilol (Ictus 6,25mg, Biolab Sanus Farmacêutica ltda, São Paulo, Brazil), O-Dianisine Sigma (São Paulo, Brazil), antibodies (Santa Cruz Biotechnology, INTERPRISE, Brazil): COX-2; RANK; RANKL; SOCS-1, Streptavidin-HRP-conjugated secondary antibody (Biocare Medical, Concord, CA, USA). TrekAvidin-HRP Label + Kit from Biocare Medical, Dako, USA. IL-1β, IL-10, TNF-α ELISA kit (R&D Systems, Minneapolis, MN, USA).

Thin colon sections (3 μm, n = 5) were taken, transferred to silanized slides (Dako, Glostrup, Denmark) and subjected to deparaffinization and hydration processes. The intestinal tissue was then washed with 0.3% Triton X-100 in phosphate buffer, treated with 3% hydrogen peroxide, and incubated overnight at 4°C with the following primary antibodies: iNOS, 1:500, p38 MAPK, 1:400, NF-κB p65, 1:100 and SOCS-1, 1:800 (Santa Cruz Biotechnology, Interprise, Brazil). After the slices were washed with phosphate buffer, they were incubated with a streptavidin-HRP-conjugated secondary antibody (Biocare Medical, Concord, CA, USA) for 30 min. Immunoreactivity was visualised with a colourimetric-based detection kit following the protocol provided by the manufacturer (TrekAvidin-HRP Label + Kit from Biocare Medical, Dako, USA) [28 ].Known positive and negative controls were included in each batch using planimetry microscopy (Olympus BX50, Morphology Department/ UFRN) with a high-powered lens (40x). Immunostaining intensity was determined, and the following scores from 1 to 4 were given: 1, absence of positive cells; 2, small number of positive cells or isolated cells; 3, moderate number of positive cells; 4, large number of positive cells. Labelling intensity was evaluated by two previously-trained examiners in a double-blind fashion. Three sections were evaluated per animal.

Tissue was processed as described previously [21 (link)]. Using a microtome, six 4-μm-thick sections of check pouch tissue were obtained from each of the Normal, 5-FU/Saline, AZT1/5-FU, and AZT5/5-FU groups. Samples were transferred to gelatine-coated slides. Each tissue section was deparaffinised, rehydrated, washed with 0.3% Triton X-100 in phosphate buffer, and quenched with endogenous peroxidase (3% hydrogen peroxide). Tissue sections were incubated overnight at 4°C with primary antibodies (Santa Cruz Biotechnology, INTERPRISE, Brazil) against VEGF, FGF, KGF, and TGF-α (all at 1:400 dilution). Slices were washed with phosphate buffer and incubated with a streptavidin/horseradish peroxidase (HRP)-conjugated secondary antibody (Biocare Medical, Concord, CA, USA) for 30 min. Immunoreactivity to TGF, KGF, FGF, and VEGF was visualised by a colorimetric-based detection kit following the manufacturer’s protocol (TrekAvidin-HRP Label + Kit from Biocare Medical, Dako, USA).

Methotrexate was purchased from LIBBs Farmacêutica Ltda, São Paulo, Brazil. Olmesartan medoximil (Benicar 20 mg, Daiichi Sankyo Brazil Farmacêutica Ltda, São Paulo, Brazil), O-Dianisine Sigma (São Paulo, Brazil), antibodies (Santa Cruz Biotechnology, INTERPRISE, Brazil): COX-2; MMP-2; MMP-9; RANK; RANKL; SOCS-1, Streptavidin-HRP-conjugated secondary antibody (Biocare Medical, Concord, CA, USA). TrekAvidin-HRP Label + Kit from Biocare Medical, Dako, USA. IL-1β, IL-10, TNF-α ELISA kit (R&D Systems, Minneapolis, MN, USA).

Thin wound skin biopsy sections (3 µm) were obtained from each group with a microtome and transferred to gelatin-coated slides. Each tissue section was then deparaffinized and rehydrated. The wound biopsy slices were washed with 0.3% Triton X-100 in phosphate buffer (PB) and quenched with endogenous peroxidase (3% hydrogen peroxide). Tissue sections were incubated overnight at 4°C with primary antibodies (Santa Cruz Biotechnology, Interprise, Santa Cruz, CA, USA) against VEGF and primary antibody (Spring-Abcam, Massachusetts, USA). Dilution tests (three dilutions) were performed with all antibodies to identify the 1:100 dilution as appropriate. Slices were washed with a phosphate buffer and incubated with a streptavidin/HRP-conjugated secondary antibody (Biocare Medical, Concord, CA, USA) for 30 min. Immunoreactivity to the various proteins was visualized with a colorimetric-based detection kit following the protocol provided by the manufacturer (TrekAvidin-HRP Label + Kit from Biocare Medical, Dako, CA, USA). Sections were counter-stained with hematoxylin. Known positive controls and negative controls were included in each sample set. Planimetry microscopy (Nikon E200 LED, Morphology Department/UFRN) with a high-power objective (×10 and ×40) was utilized to score the intensity of cell immunostaining, according to the methodology used by Araújo Júnior et al. (2014 (link)).