20 μm thick brain sections were cut on a cryostat. Brain sections (free float) were incubated overnight with the following antibodies: rpS6 (1:500) and phospho (Ser 240 and 244) rpS6 (1:500). The sections were subsequently incubated with a biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA,) at 1:200 for 1 hr and then incubated with an avidin-biotin peroxidase complex solution (Vector Laboratories, Burlingame, CA) at 1:100 for 1hr. After each incubation, the sections were rinsed for 15 min with PBS containing 0.3% Triton X-100. Finally, the products of the immunoreaction were visualized using diaminobenzidine (DAB) as the substrate (Vector Laboratories, Burlingame, CA). To evaluate the ischemic core, the transition area and peri-infarct area, sections stained with cresyl violet were compared to those that were immunostained.
Avidin biotin peroxidase complex solution
Manufactured by Vector Laboratories
Sourced in United States
The Avidin-biotin-peroxidase complex solution is a pre-formed complex composed of avidin, biotin, and horseradish peroxidase. This solution can be used as a detection system in various immunohistochemical and enzyme-linked immunosorbent assay (ELISA) applications.
Automatically generated - may contain errors
Lab products found in correlation
Permount, by Merck Group (9 mentions)
Bx51 light microscope, by Olympus (8 mentions)
Diaminobenzidine, by Merck Group (5 mentions)
Abc kit, by Vector Laboratories (3 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (2 mentions)
Rabbit anti nrsf rest antiserum, by Fortis Life Sciences (2 mentions)
Normal horse or goat serum, by Vector Laboratories (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Diaminobenzidine dab, by Vector Laboratories (1 mentions)
Biotinylated anti mouse igg, by Vector Laboratories (1 mentions)
Biotinylated goat anti rabbit igg, by Merck Group (1 mentions)
Vectastain elite, by Vector Laboratories (1 mentions)
Diaminobenzidine, by Vector Laboratories (1 mentions)
Diaminobenzidine dab, by Merck Group (1 mentions)
Bx51 light microscopy, by Olympus (1 mentions)
Diaminobenzidine substrate kit, by Vector Laboratories (1 mentions)
Tnf α, by Abcam (1 mentions)
Ly6b 2 clone 7 4, by Bio-Rad (1 mentions)
Icam 1, by Sino Biological (1 mentions)
Bx50 microscope, by Olympus (1 mentions)
21 protocols using avidin biotin peroxidase complex solution
1
Immunohistochemical Analysis of Phosphorylated rpS6
2
Immunohistochemical Detection of GFP/eYFP
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Following rinses in hydrogen peroxide (2% in PB, 15 min) and Triton X-100 (2% in PB, 30 min), sections were incubated in a purified rabbit antiserum against GFP that also recognizes eYFP (1:500; EXBIO, Prague, Czech Republic) + 2% Triton X-100 + 3% normal goat serum (NGS) + 1% bovine serum albumin (BSA) in PB (16 h, 20°C). After several rinses in PB, sections were incubated in biotinylated goat anti-rabbit IgG (1:100; Sigma–Aldrich, St. Louis, MO, USA) + 2% Triton X-100 + 3% NGS + 1% BSA in PB (2 h, 20°C). Following new rinses in PB, the sections were incubated in avidin-biotin-peroxidase complex solution (1:100; Vectastain Elite, Vector Laboratories, Burlingame, CA, USA) + 2% Triton X-100 (4°C, 16 h). Peroxidase activity was visualized using a glucose oxidase-DAB-nickel protocol (Shu et al., 1988 (link)). Sections, were lightly counterstained with Thionin for cytoarchitectonic reference, defattened, dehydrated and coverslippped with DePeX.
3
Immunohistochemical Analysis of Tissue Sections
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Free-floating brain, deparaffinized liver, adipose tissue, and pancreatic sections were incubated with primary antibodies that were diluted in blocking solution overnight at 4 °C (Supplementary Table 1 ). After three washes with 0.1 M PBS, sections were incubated for 1 h at room temperature with biotinylated secondary antibody. After washing, sections were incubated in an avidin-biotin-peroxidase complex solution (Vector Laboratories, CA, USA) and developed with diaminobenzidine (Vector Labs) containing 0.025% H2O2. Sections were then dehydrated through graded alcohol solutions, cleared in xylene, and mounted under a coverslip with Permount (Sigma-Aldrich, St. Louis, MO, USA). Image of the stained sections were captured using a BX51 light microscope (Olympus). Immunohistochemical intensity data for insulin, C-peptide, TonEBP, HMGB1, and iba-1 were obtained from selected images using i-Solution (IMT i-Solution Inc., Vancouver, BC, Canada). Three fields (200 × 200 µm2) were randomly selected on each section from two continuous sections (n = 4 mice per group). Intensity measurements are represented as the percentage of the mean number of pixels versus the corresponding value at which the pixel of the respective intensity was present.
4
Immunohistochemical Localization of NRSF
5
Immunohistochemical Analysis of TonEBP
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Deparaffinized epididymal fat pads and frozen-brain sections were incubated overnight at 4 °C in a humidified chamber with primary antibodies (Additional file 1 : Table S1) diluted in blocking serum. After washing, sections were incubated in avidin–biotin–peroxidase complex solution (Vector Laboratories, Burlingame, CA, USA). Sections were developed with 0.05% diaminobenzidine (DAB, Sigma-Aldrich) containing 0.05% H2O2 and were dehydrated through graded alcohols, cleared in xylene, and coverslipped with Permount (Sigma-Aldrich). Sections were visualized using BX51 light microscopy (Olympus). For the measurement of the intensity of immunostained TonEBP from brain sections, three fields (200 × 200 µm2) were randomly selected from each section (n = 3–4 per group).
6
Immunohistochemical Analysis of Skeletal Muscle
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Sections of deparaffinized skeletal muscle were incubated in a solution of 0.3% H2O2 for 30 min. After washing, sections were blocked by incubation for 1 h at room temperature in donkey serum. The sections were then incubated at 4 °C overnight with primary antibodies (Table S2 ). After washing three times with 0.1 M PBS, the sections were then incubated for 1 h at room temperature with a secondary biotinylated antibody (1:200). After washing, the sections were incubated in avidin–biotin–peroxidase complex solution (Vector Laboratories) and developed using 0.05% DAB/horseradish peroxidase substrate (Vector Laboratories). The sections were then dehydrated using a graded alcohol series, cleared using xylene, mounted under a coverslip, and observed under a BX51 light microscope (Olympus). Immunohistochemical data for F4/80 and ferritin were obtained from selected images. Six fields (50 × 50 μm2) were randomly selected on each section using iSolution software (IMT iSolution Inc.) by an observer unaware of the group assignments.
7
Immunohistochemical Analysis of Kidney Tissue
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After fixation, kidney tissues were paraffin-embedded and cut into 5-µm-thick longitudinal sections. After deparaffinization, the slide samples were incubated with appropriate primary antibodies as follows. Antibodies against calcineurin (BD Bioscience, San Jose, CA, USA), neutrophil infiltration protein Ly6B.2 (clone 7/4, AbD Serotec, San Diego, CA, USA), TNF-α (Abcam, Cambridge, UK), caspase-3 (Asp175, Cell Signaling Technology, Danvers, MA, USA), NFATc1 (NFAT2, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and intercellular adhesion molecule-1 (ICAM-1; Sino Biological Inc., Beijing, China) were used. Sections were incubated with primary antibody at 4℃ overnight. After washing three times with 0.1 M phosphate buffered saline (PBS), sections were incubated with the biotin tagged an appropriate secondary antibody at room temperature (RT) for 1 hour. After washing three times with 0.1 M PBS, sections were incubated in avidin-biotin-peroxidase complex solution (Vector Laboratories, Inc., Burlingame, CA, USA) and developed with diaminobenzidine substrate kit (Vector Laboratories, Inc.). After dehydration, sections were mounted and visualized with a BX50 microscope (Olympus, Tokyo, Japan), and digital images were captured. Captures were randomly distributed on 5 areas of all field and represented as high power field using Image J.
8
Immunohistochemistry of Liver Sections
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Deparaffinized sections of liver were placed in 0.3% H2O2 for 10 minutes, washed, and incubated in blocking serum for 20 min. Sections were incubated in primary antibodies (Table S1 ) at 4 °C overnight and with a secondary biotinylated antibody for 1 h at room temperature. After washing, sections were incubated in an avidin-biotin-peroxidase complex solution (Vector Laboratories, Burlingame, CA, USA) and developed with 0.05% diaminobenzidine (Sigma-Aldrich) containing 0.05% H2O2. The sections were then dehydrated in graded alcohols, cleared in xylene, and mounted under a coverslip with Permount (Sigma-Aldrich). Sections were visualized under a BX51 light microscope (Olympus).
9
Immunohistochemical Analysis of Insulin in Pancreatic Tissue
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The pancreases were fixed in the 10% neutral buffered formalin processed routinely and embedded in paraffin wax. The sections of pancreas were cut at 4 μm thick. The sections were stained with hematoxylin and eosin (H&E). For IHC, the sections were incubated in a solution of 3% hydrogen peroxide prepared in methanol for 40 min and microwaved at 750 W for 10 min in 0.01 mol/L citrate buffer. After being blocked with rabbit serum (Vector Laboratories, Burlingame, CA, USA) for 30 min, the tissue sections were immunostained with the primary antibody, a rabbit monoclonal anti-insulin (1 : 200, Santa Cruz Biotechnology, USA). The antigen-antibody complex was visualized by an avidin-biotin peroxidase complex solution using an ABC kit (Vector Laboratories, Burlingame, CA, USA). The sections were rinsed in distilled water and counterstained with Mayer's hematoxylin.
10
Immunohistochemical Quantification of CD68+ Cells
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The deparaffinized sections of liver was placed in 0.3% H2O2 for 10 minutes, washed, and incubated in blocking serum for 20 min. Sections were incubated in primary antibodies (Supplementary Table 1 ) at 4℃ overnight and with a secondary biotinylated antibody for 1 h at room temperature. After washing, the sections were incubated in avidin-biotin-peroxidase complex solution (Vector Laboratories, Burlingame, CA, USA) and developed with 0.05% diaminobenzidine (Sigma-Aldrich) containing 0.05% H2O2. The sections were then dehydrated in graded alcohols, cleared in xylene, and mounted under a coverslip with Permount (Sigma-Aldrich). The CD68-positive immunostained cells were counted from three sections (n=3~4 per each group) by observers blinded to the treatment conditions using 200x objectives. The sections were visualized under a BX51 light microscope (Olympus) and the quantitative analysis was performed using analySIS-FIVE program (Olympus Soft Imaging System).
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