5-μm esophageal paraffin tissue sections of mouse esophagus were immunostained with antiserum against mouse eosinophil major basic protein (anti-MBP) as per the method described previously. 25 (link)–27 (link) In brief, endogenous peroxidase. In. the. tissues was quenched with 0.3% hydrogen peroxide in methanol followed by non-specific protein blocking using 3 % normal goat serum. Tissue sections were then incubated with rat anti-MBP (1:6000 dilution) overnight at 4°C (purchased from Mayo Clinic, Scottsdale, AZ, USA), followed by a 1:250 dilution of biotinylated goat anti-rat IgG secondary antibody and avidin-peroxidase complex (Vector Laboratories, Burlingame, CA) for 30 minutes each. Slides were further developed with nickel diaminobenzidine-cobalt chloride solution to form a black precipitate, and counterstained with nuclear fast red. Negative controls included replacing the primary antibody with normal rabbit serum to control for endogenous biotin and peroxidase activity. Eosinophils were quantified by counting the anti-MBP positive-stained cells in each tissue section with the assistance of digital morphometry using the Metamorph Imaging System (Universal Imaging Corp, West Chester, PA) and expressed as eosinophils/mm2 tissue area as described earlier. 28 (link), 29 (link)
Avidin peroxidase complex
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
The Avidin-peroxidase complex is a laboratory reagent used in various biochemical and immunological applications. It consists of the protein avidin conjugated with the enzyme peroxidase. The core function of this complex is to act as a signal amplification system, allowing for the detection and visualization of target molecules in biological samples.
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Lab products found in correlation
Biotinylated mouse secondary antibody, by Vector Laboratories (3 mentions)
Brdu specific mouse monoclonal antibody, by Roche (3 mentions)
Permount, by Thermo Fisher Scientific (3 mentions)
Biotinylated goat anti rat igg secondary antibody, by Vector Laboratories (2 mentions)
Metamorph imaging system, by Universal Imaging (2 mentions)
Biotinylated anti rat igg secondary antibody, by Vector Laboratories (2 mentions)
Biotinylated goat secondary antibody, by Vector Laboratories (2 mentions)
Goat anti dcx antibody, by Santa Cruz Biotechnology (2 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (2 mentions)
3 3 diaminobenzidine (dab), by Thermo Fisher Scientific (1 mentions)
Biotin conjugated secondary antibody, by Vector Laboratories (1 mentions)
Ab8536, by Abcam (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Marinol, by Muto Pure Chemicals (1 mentions)
Ab8940, by Abcam (1 mentions)
Masson trichrome, by Merck Group (1 mentions)
Jc70a, by Agilent Technologies (1 mentions)
Aquamount medium, by Avantor (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Avidin and biotin blocking solution, by Vector Laboratories (1 mentions)
21 protocols using avidin peroxidase complex
1
Quantification of Eosinophils in Mouse Esophagus
2
Tyrosine Hydroxylase Immunohistochemistry
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Every third slice containing a graft was selected for tyrosine hydroxylase (TH) immunohistochemistry. After 3 rinses in PBS, tissue sections were preincubated in 0.3% Triton X-100 in PBS plus 10% horse serum (HS) for 60 min, washed, and incubated overnight with the rabbit polyclonal anti-TH antibody (1:500; Pel Freez) for 48 h at 4 °C in 0.1% Triton X-100 in PBS plus 2.5% HS. Following 3 washes, sections were incubated with a biotinylated secondary antibody (horse antirabbit 1:200; Vector Labs) in 0.1% Triton X-100 in PBS plus 2.5% HS for 90 min. Endogenous peroxidase was blocked by 3% H2O2 and 10% methanol in PBS for 10 min. Following incubation with an avidin–peroxidase complex (1:150; Vector Labs) for 45 min, specifically bound antibody was visualized with a metal-enhanced 3,3′-diaminobenzidine (DAB) substrate kit (Pierce). Sections were dehydrated in alcohol, cleared in xylene, and mounted in Eukitt.
3
Immunohistochemistry for BrdU-Positive Cells
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Immunohistochemistry for the BrdU-positive cells in the hippocampal dentate gyrus was performed, as the previously described method (Jung and Kim, 2017 (link); Kim et al., 2017 ). The sections were permeabilized by incubating in 0.5% Triton X-100 in PBS for 20 min, then they were pretreated in 50% formamide-2×standard saline citrate at 65°C for 2 hr, denaturated in 2 N HCl at 37°C for 30 min, and they were rinsed twice in 100 mM sodium borate (pH, 8.5). Afterwards, the sections were incubated overnight at 4°C with BrdU-specific mouse monoclonal antibody (1:600; Roche, Mannheim, Germany). The sections were then washed three times with PBS and incubated for 1 hr with the biotinylated mouse secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA). The sections were incubated for another 1 hr with avidin-peroxidase complex (1:100; Vector Laboratories). For visualization, the sections were incubated for 5 min in 50 mM Tris-HCl (pH, 7.6) containing 0.02% 3,3′-diaminobenzidine (DAB), 40 mg/mL nickel chloride, and 0.03% hydrogen peroxide. The sections were finally mounted onto gelatin-coated slides. The slides were air-dried overnight at room temperature, and the coverslips were mounted using Permount (Thermo Fisher Scientific Inc., FairLawn, NJ, USA).
4
BrdU Immunohistochemistry Staining Protocol
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BrdU immunohistochemistry was done, as mentioned in the below (Ji et al., 2020 (link)). After the sections were treated with 0.5% Triton X-100 in PBS for 20 min, incubated with 50% formamide-2 x standard saline citrate at 65°C for 2 hr, denatured in 2 N HCl for 30 min at 37°C, and washed twice in 100 mM sodium borate (pH, 8.5). Afterwards, the sections were incubated with BrdU-specific mouse monoclonal antibody (1:600; Roche, Mannheim, Germany) overnight at 4°C. After washing 3 times with PBS, the sections were treated with biotinylated mouse secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA) for 1 hr. The sections were treated with an avidin-peroxidase complex (1:100; Vector Laboratories) for another 1 hr, treated with 50 mM Tris-HCl (pH, 7.6) containing 0.02% diaminobenzide, 40-mg/mL nickel chloride, and 0.03% H2O2 in 50 mM Tris-HCL (pH, 7.6) for 5 min. BrdU-positive cells were confirmed by treating with a mouse antineuronal nuclear antibody (1:1,000; Chemicon International, Temecula, CA, USA). After washing 3 times with PBS, the sections were treated with a biotinylated anti-mouse secondary antibody for 1 hr, and then treated with a reaction mixture of 0.03% diaminobenzide with 0.03% H2O2 for 5 min for staining.
5
Immunohistochemical Analysis of Murine Lungs
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The lungs of all mice were inflation fixed with formalin and embedded in paraffin for immunohistochemical staining. Sections 5 μm thick were taken at 500 μm intervals from a random starting point, and were deparaffinised and rehydrated in xylene and graded ethanol. Antigen retrieval was performed using 10 mM citrate buffer (pH 7.0) in a pressure cooker at 120°C. Sections were permeabilised in TBS-Tween for 30 minutes before blocking of endogenous peroxidise activity with 3% H2O2 for 10 minutes. After rinsing in TBS-Tween, slides were blocked in 10% (v/v) normal goat serum for 30 minutes. HRV-1B antisera (American Type Culture Collection, Manassas, VA, USA) was diluted to 1:500 in blocking buffer and added to the slides overnight at 4°C, followed by incubation with biotinylated goat anti-rabbit IgGs (1:500) and avidin-peroxidase complex (Vector Laboratories, Peterborough, UK). Finally sections were developed with diaminobenzidine substrate and counterstained with 10% haematoxylin. Images were visualized at 20× magnification under light microscopy using an Olympus BX43 System Microscope (Olympus Australia Pty. Ltd., Mount Waverely, Australia).
6
Quantification of Eosinophil Major Basic Protein
7
Quantifying Rabbit Cornea Leukocytes
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Rabbit corneas were fixed for 24 h in 3.7% formaldehyde and then paraffin-embedded. Three-μm thick sections were incubated with primary antibody anti rabbit-CD45 (Antigenix America) for 1h and then incubated with biotin-conjugated secondary antibody (1:100, Vector Labs, Burlingame, CA, USA) for 1h. Samples were then incubated with avidin-peroxidase complex (Vector), and reveled with DAB (Invitrogen) enhanced or not with Cl2Co. Leukocyte infiltration was measured by counting CD45+ cells in an area of 0,66 mm2 under a 40x objective and compared between groups.
8
Immunohistochemical Detection of DCX
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DCX immunohistochemistry was assessed using a previously described method (Shin et al., 2013 (link)). The sections were treated with 3% H2O2 for 30 min at room temperature. Next, the sections were blocked with 10% normal rabbit serum in PBS with 1% bovine serum albumin and 0.2% Triton X-100 for 1 hr and incubated with goat anti-DCX antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. After washing 3 times, the sections were incubated with biotinylated goat secondary antibody (1:200; Vector Laboratories) for 1 hr, followed by incubation for 1 hr with avidin-peroxidase complex (1:100; Vector Laboratories). For immunostaining, the sections were visualized in 50-mM Tris–HCl (pH, 7.6) containing 0.03% DAB and 0.03% H2O2. Then, the tissue samples were rinsed with PBS three times and mounted on gelatin-coated slides. The slides were dehydrated through alcohol and coverslipped with Permount (Fisher Scientific Inc.).
9
Doublecortin Immunohistochemistry Protocol
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Following a method described elsewhere [18 (link)], doublecortin (DCX) immunohistochemistry was performed. After the sections were incubated in 3% H2O2 for 30 minutes at room temperature, they were blocked by 10% normal rabbit serum in PBS with 0.2% Triton X-100 and 1% bovine serum albumin for 1 hour. The section was incubated in goat anti-DCX antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4℃ overnight. After washing 3 times, the sections were treated with a biotinylated goat secondary antibody (1:200; Vector Laboratories) for 1 hour, followed by another hour with avidin–peroxidase complex (1:100; Vector Laboratories). The sections were visualized in 50mM Tris-HCl (pH, 7.6) containing 0.03% hydrogen peroxide and 0.03% DAB for immunostaining. The slides were dehydrated by alcohol and mounted using Permount coverslips (Thermo Fisher Scientific Inc.).
10
Immunohistochemical Detection of Eosinophil Markers
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