Colon sections (3 μm; n = 5 fields per slide/3 per group) were obtained and transferred to silanized slides (Dako, Glostrup, Denmark), dewaxed, and hydrated. Slides were washed with 0.3% Triton X-100 in phosphate buffer, treated with 3% hydrogen peroxide, and incubated overnight at 4 °C with primary antibodies for mucin-type 2 (MUC-2, 1:200) and zonula occludens 1 (ZO-1, 1:200) (Santa Cruz Biotechnology, Interprise, São Paulo, Brazil). After washing, slides were incubated with secondary streptavidin–HRP-conjugated antibody (1:500, Biocare Medical, Concord, CA, USA) for 30 min, and immunoactivity for MUC-2 and ZO-1 was carried out (Trek Avidin-HRP label + Biocare Medical, CA, USA). Samples were visualized under an optical microscope (Leica DM750, Schweiz, Switzerland) with Qwin system coupled to a camera (Leica ICC50 HD, Wetzlar, Germany). The intensity of immunostaining was determined for each animal using 5 random fields (real area 327.68 × 245.76 μm). Quantification was performed by AVSoft Bioview version 4.0.1.
Trekavidin hrp label
Manufactured by Biocare Medical
Sourced in United States
TrekAvidin-HRP Label is a conjugate of avidin and horseradish peroxidase (HRP). Avidin has a high affinity for biotin, and the HRP enzyme can be used as a reporter molecule in various bioassays and detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Silanized slides, by Agilent Technologies (1 mentions)
Dm750, by Leica camera (1 mentions)
Icc50 hd, by Leica camera (1 mentions)
K3468, by Agilent Technologies (1 mentions)
Trekkie universal link, by Biocare Medical (1 mentions)
Pdgfr β, by ABclonal (1 mentions)
Mcp 1, by Abcam (1 mentions)
α sma, by Merck Group (1 mentions)
Ab16901, by Abcam (1 mentions)
Ab4051, by Abcam (1 mentions)
3 3 diaminobenzidine (dab), by Biocare Medical (1 mentions)
5 protocols using trekavidin hrp label
1
Immunohistochemical Analysis of Intestinal Mucin and Tight Junctions
2
Immunohistochemical Detection of E-Cadherin
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Paraffin-embedded tissues were sectioned (3 μm) and collected in series on glass slides coated with 2% 3-aminopropyltriethsilane (Sigma-Aldrich, St Louis, MO, USA). Following deparaffinization by immersion in xylene, the sections were immersed in alcohol and incubated with 3% hydrogen peroxide for 40 min. To retrieve antigens, the sections were immersed in citrate buffer (pH 6.0) for 20 min. Afterwards, the sections were incubated for 20 min with 3% normal goat serum at room temperature. The slides were incubated at 4°C overnight with the primary antibody monoclonal mouse anti-E-Cadherin human (SPM471, Spring Bioscience, Pleasanton, CA, USA) at 1:200 in a humidifier. After washing with Phophate Buffered Saline, the sections were labeled with TrekAvidin-HRP Label (STHRP700 L10; Biocare Medical, Concord, CA, USA) and then incubated with 3,3’-diaminobenzidine (K3468; DAKO, Glostrup, Denmark) for 2–5 min at room temperature. The sections were then stained with Mayer’s hematoxylin and covered (Entellan-Mikroskopie-Merck, Darmstadt, Germany). Negative controls were obtained through the omission of primary antibody, and normal oral mucosa samples with known positive reactivity were included as positive controls.
3
Immunohistochemical Analysis of Periodontal Tissue
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Only the controls (Sham and positive control) and the experimental group (PLGA + 10 mg/kg met) were included in this step. Fine sections of periodontal tissue (4 µm) (3 mandibles per group) were produced using a microtome and transferred onto gelatin coated slides. Each section was deparaffinized and rehydrated. Gingival and periodontal tissues were washed with 0.3% Triton X-100 in phosphate buffer, then extinguished with peroxidase (3% hydrogen peroxide) and incubated with the following primary antibodies (Santa Cruz Biotechnology, Interprise, Brazil) overnight at 4 °C: RANKL, 1400; OPG, 1400; cathepsin K, 1400; and osteocalcin, 1400, which were washed with phosphate buffer and incubated with streptavidin-HRP-conjugated secondary antibodies (Biocare physicians, Concord, CA, USA) for 30 min, and immunoreactivity for RANK, RANK-L, OPG, cathepsin K, and osteocalcin were visualized using a colorimetric detection kit following the manufacturer’s instructions (TrekAvidin-HRP Label, Biocare Medical, Pacheco, CA, USA; TrekAvidin-HRP Kit, Dako, Carpinteria, CA, USA).
4
Quantification of Kidney Fibrosis Markers
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The kidney tissue was embedded in 4-μm thick paraffin sections. Then, it was deparaffinized and rehydrated using xylene and alcohol series. The specimens were stained with SR to measure the fraction area of interstitial fibrosis and with periodic acid-Schiff (PAS) to determine tubular injury. This step was followed by antigen retrieval, blocking peroxidase using 3% H2O2 in PBS solution, and blocking non-specific antigen using Background Sniper for immunostaining. The slides were incubated with α-SMA (1:400 dilution, Sigma, Cat. No. A2547) and CD68 (1:400 dilution, Abcam, Cat. No. ab955), PDGFR-β (1:200 dilution, Abclonal, Cat. No. A2180), MCP-1 (1:100 dilution, Abcam, Cat. No. ab25124) as the primary antibodies; TrekAvidin-HRP label, conjugated to anti-rabbit Trekkie universal Link (Biocare Medical®), as the secondary antibody; and diaminobenzidine tetrahydrochloride (DAB). The α-SMA immunostaining was applied to measure myofibroblast expansion, and CD68 antibody was used for counting macrophage cells. The quantification was performed from 15 fields for each sample at ×400 magnification, using ImageJ software.
5
Immunohistochemical Analysis of Mammary Tumors
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At the end of the treatment, all rats were euthanized and the mammary tumors were fixed in 10% neutral buffered formalin. Paraffin blocks were made and cut into 5 μm slices for each rat. Three consecutive slices were stained immunohistochemically. Slides were incubated in antigen retrieved (pH 6.0), for 15 min at 95°C, cooled at 27ºC (room temperature/RT) (for caspase-3 antibody), followed by washing with PBS. Slides were then incubated with endogenous activity blocking H2O2 3% (Biocare Medical, California, USA) (HER2 and caspase-3 antibody) for 30 min at RT. Next, slides were incubated with primary antibody against HER2 (1:100; mouse monoclonal [3B5] antibody to ErbB2, ab16901; Abcam, Cambridge, UK) and caspase-3 (1:250; rabbit polyclonal antibody to caspase-3, ab4051; Abcam) at 4°C overnight. Slides were incubated with a secondary antibody (Trekkit Universal Link; Biocare Medical) for 30 min at RT and were then incubated with Trek Avidin-HRP Label (Biocare Medical) for 30 min at RT. Following rinsing with PBS, visualization was performed using the peroxidase substrate 3,3-diaminobenzidine (Biocare Medical) as the chromogen. Then, counterstaining with hematoxylin was carried out, followed by mounting. Apart from the palpation method, the presence of a tumor mass was also confirmed through hematoxylin-eosin and Masson’s trichrome staining.
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