The obtained 4 μm paraffin-embedded sections (n = 3 per group) were deparaffinized using xylene and rehydrated with EtOH (100%, 90%, 80%, and 70%), followed by treatment with an endogenous peroxidase blocker, and finally washed with PBS. These sections were incubated with the primary antibody, BDNF (dilution 1 : 500), at 4°C. After washing in PBS, each section was incubated with a biotinylated anti-goat and anti-rabbit IgG (dilution 1 : 200) for 1 h and then with avidin-biotin horseradish peroxidase complex (Vector Laboratories, CA, USA). The optical density of BDNF immunoreactivity in the CA1 and CA2/3 regions was analyzed using ImageJ software. The images were photographed at 4–40x magnification using a microscope (Olympus).
Avidin biotin horseradish peroxidase complex
Manufactured by Vector Laboratories
Sourced in United States, Canada
The Avidin-biotin horseradish peroxidase complex is a reagent used in various biological and biochemical applications. It consists of the protein avidin, which binds to the small molecule biotin, and the enzyme horseradish peroxidase. This complex can be used to detect and visualize the presence of biotinylated molecules in a sample.
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Lab products found in correlation
Biotinylated anti mouse igg or anti rabbit igg secondary antibodies, by Vector Laboratories (2 mentions)
Bz 9000 series, by Keyence (2 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (2 mentions)
Dab kit, by Vector Laboratories (2 mentions)
Osteocalcin, by Takara Bio (1 mentions)
Nf κb p65, by Rockland Immunochemicals (1 mentions)
Tyramide signal amplification, by PerkinElmer (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
Alexa 546, by Thermo Fisher Scientific (1 mentions)
Nis elements ar software, by Nikon (1 mentions)
3 3 diaminobenzidine substrate, by Vector Laboratories (1 mentions)
Bx40 microscope, by Olympus (1 mentions)
Rabbit anti bdnf antibody, by Santa Cruz Biotechnology (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Mayer s hematoxylin, by Merck Group (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Citric acid based antigen unmasking solution, by Vector Laboratories (1 mentions)
Novared chromagen, by Vector Laboratories (1 mentions)
Biotinylated goat anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
Dab kit, by Agilent Technologies (1 mentions)
28 protocols using avidin biotin horseradish peroxidase complex
1
Quantifying BDNF Immunoreactivity in Hippocampal Regions
2
Osteocalcin and NF-κB-p65 Immunofluorescence
3
Immunohistochemical Analysis of Lysyl Oxidase in Tibia
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To determine the relative distribution of LOX, immunohistochemical analyses were performed within the tibia paraffin sections of SHAM, OVX, and ALF-treated groups (see PSR staining, above). The endogenous peroxidase activity was quenched by incubating the slides in 0.3% H2O2 in methanol for 30 min. The nonspecific binding was blocked using normal goat serum for 20 min. The sections were incubated overnight at 4°C with primary antibody against LOX (IMGENEX, San Diego, CA), which was diluted 1:200 in PBS. The specificity of immunoreactivity was confirmed by using normal rabbit serum as negative controls. The sections were incubated with biotinylated rabbit IgG (Vector Laboratories, Inc., Burlingame, CA) and then with avidin-biotin-horseradish peroxidase complex (Vector Laboratories, Inc.). After several washes with PBS, the sections were incubated with 3, 3′-diaminobenzidine substrate (Vector Laboratories, Inc.) to visualize the immunoreactivity. The sections were observed under light microscopy (BX40 microscope, Olympus Co.). The area observed was 1.0–2.0 mm below the tibial growth plate and 200 µm thickness from the bone marrow side of cortical bone. LOX expression was evaluated by counting the number of immunopositive osteocytes/area. Data were collected from randomly selected four sections per tibial bone from two animals in each group.29 (link)
4
BDNF Expression in DRG Neurons
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Rats were anesthetized deeply with ether and perfused transcardially with 50 mL of saline, followed by 500 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The L3, L4, and L5 DRG were dissected and post-fixed in the same fixative for 30 minutes, incubated in phosphate-buffered 20% sucrose solution overnight. These tissues were embedded in OCT compound (Tissue-tek®; Sakura Finetek, Tokyo, Japan) and 8 μm thick frozen sections were cut. For visualization of BDNF in the DRG, the avidin-biotin-horseradish peroxidase complex method was used. These sections were incubated overnight with rabbit anti-BDNF antibody (1:4000, #sc-546; Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by incubation with biotinylated goat anti-rabbit IgG (immunoglobulin G) (1:200) and avidin-biotin-horseradish peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Immunoreaction products were visualized with diaminobenzidine and nickel ammonium sulfate. DRG neurons were categorized as small (<20 μm diameter), medium (20–40 μm diameter), and large (>40 μm diameter), and the proportion of BDNF-immunoreactive (IR) neurons was analyzed in each group. The cell counting was performed by a blinded observer. Three representative sections that contained over 70 neurons, total at least 210 each neuron, with distinct nuclei were randomly selected in each of the DRG.
5
Immunoperoxidase Staining Protocol
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The immunoperoxidase staining method was modified from the avidin–biotin complex technique as described previously.17 In brief, slides (5 μm) were deparaffinized. After antigen retrieval, the slides were digested in 0.05% trypsin. The endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide, and the slides were then treated with 10% normal goat for 30 min at RT. After overnight incubation with primary antibodies (a) Cleaved Caspase‐3 (1:100 dilution; Cell Signaling Technology); (b) Ki67 (1:100 dilution; Cell Signaling Technology), the slides were incubated with biotinylated secondary antibodies and subsequently with avidin–biotin–horseradish peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Antibody detection was performed with the 0.125% aminoethyl carbazole chromogen substrate solution (AEC substrate) from Sigma (St. Louis, MO, USA). After counterstaining with Mayer's hematoxylin (Sigma (St. Louis, MO, USA)), the sides were mounted and imaged.
6
Immunohistochemical Staining Protocol
7
Immunohistochemical Analysis of Breast Cancer
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Sections frozen in OCT were fixed in acetone and chloroform. After overnight incubation with primary antibodies, including mouse monoclonal anti-HER-2, anti-YB-1, and anti-E-cadherin, the slides were washed again with Tris-buffered saline and Tween20 and incubated with biotinylated secondary antibodies and subsequently with the avidin–biotin–horseradish peroxidase complex (Vector Laboratories, San Mateo, CA, USA). Antibody detection was performed using 3,3′-diaminobenzidine, and the tissue sections were counterstained with Mayer's hematoxylin, washed, mounted with Universal Mount, and dried on a 56°C hot plate. The prepared slides were examined through light microscopy.
8
Immunohistochemical Analysis of MMPs and TIMPs
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Microtome sections (6 μm thick) were deparaffinized in xylene and rehydrated by passing through graded alcohols. After washing with TBST buffer (Tris buffer saline + 0.1 % Tween 20), slices were incubated in citric buffer (pH = 6.0, 75 °C, 20 min) for heat-induced antigen retrieval. Endogenous peroxidase activity was quenched in 3 % H2O2 in methanol (10 min). Nonspecific binding of the secondary antibody was blocked by incubation with 5 % (v/v) normal goat serum in TBST (RT, 40 min). Sections were then incubated overnight with primary rabbit polyclonal antibodies against MMPs and TIMPs as described for western blots above. After rinsing with TBST, sections were incubated (1.5 h) with biotinylated goat anti-rabbit antibody (1:300) followed by avidin-biotin-horseradish peroxidase complex (Vector Laboratories, Burlingame, CA, USA) (30 min). The color reaction was developed by incubation with diaminobenzidine (DAB) and H2O2 solution. Non-specific staining was demonstrated by replacement of the primary antibody with TBST. Slides were examined under a light microscope (Jena Zeiss, Germany).
9
Immunohistochemical Localization of GPER
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The localization of GPER in the initial segment (IS), caput, corpus and cauda tissues was investigated by immunohistochemistry using a protocol similar to one previously described [3 (link)]. Tissue was paraffin embedded and sectioned at a thickness of 5 μm. Antigen retrieval was performed by submerging slides in a citric acid based antigen unmasking solution (Vector Laboratories, Inc., Burlingame CA) in Coplin jars and steam heated to 93 °C for 5 min after which they were allowed to cool to room temperature. Endogenous peroxidase activity was blocked by incubation in 0.3 % hydrogen peroxide in methanol for 30 minutes. After a blocking step, tissues were incubated for two hours at room temperature with rabbit anti-human GPR30 (1:2500; sc-48525; Santa Cruz Biotechnology). Following primary antibody incubation, sections were incubated with goat anti-rabbit biotinylated secondary antibody followed by an avidin–biotin horseradish peroxidase complex (Vector Laboratories). Immunostaining was visualized using NovaRed chromagen (Vector Laboratories). Pre-absorbed primary antibodies were substituted for primary antibody as a negative control; all other steps were identical including exposure times with NovaRed. All slides were counterstained with Immunomaster Hematoxylin and evaluated by light microscopy.
10
Immunohistochemistry for Tumor Angiogenesis
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Frozen tumor sections (7 μm) were fixed with methanol. The sections were then treated for 30 min with hydrogen peroxide (0.3%) to block endogenous peroxidase activity. After incubation with normal goat serum 15%, the sections were incubated with anti-mouse CD31 (1:100; BD Pharmigen, San Jose, CA, USA) for 1 hour at room temperature. The primary antibodies were detected using anti-rat secondary antibodies and avidin/biotin/horseradish peroxidase complex (Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. The labeled antigens were visualized with the DAB kit (DAKO Cytomation, Kyoto, Japan). The sections were counterstained with hematoxylin and examined using a BH-2 microscope (Olympus) equipped with an INFINITY1 2.0 megapixel CMOS digital camera (Lumenera Corporation, Ottawa, Canada). All images were acquired using INFINITY ANALYZE software (Lumenera Corporation) without post-acquisition processing. MVD was determined by counting three fields at ×100 magnification of the highest vascular density.
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