The DAB staining was adapted from previously described work (Kohman et al., 2012 ). Briefly, free floating sections were washed in Tris-buffering solution (TBS) and treated with 0.6% hydrogen peroxide solution for 30 min. Next, sections were placed in 50% de-ionized formamide for 90 min to denature DNA. Sections were then placed in a 10% 20x saline sodium citrate buffer for 15 min, 2N hydrochloric acid for 30 min at 37°C, and then 0.1 M boric acid (pH 8.5) for 10 min. After rinsing, sections were blocked with a solution of 0.3% Triton-X and 3% goat serum in TBS (TBS-X) for 30 min. The sections were then incubated with the primary antibody rat anti-BrdU (1:200; AbD Serotec, Raleigh, NC, USA) in TBS-X for 72 h at 4°C. Sections were then rinsed with TBS, blocked with TBS-X for 30 min, and then incubated with a biotinylated goat anti-rat secondary antibody (1:250, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 100 min. The ABC system (Vector, Burlingame, CA, USA) and diaminobenzidine kit (DAB; Sigma, St. Louis, MO, USA) were used for the chromogen.
Diaminobenzidine dab kit
Manufactured by Merck Group
Sourced in United States
The Diaminobenzidine (DAB) kit is a laboratory product manufactured by Merck Group. It is a chromogenic substrate used in various immunohistochemical and immunocytochemical applications to detect the presence of target proteins or antigens in biological samples. The kit provides a reliable and consistent way to visualize the localization of specific proteins or molecules within cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Rat anti brdu, by Bio-Rad (2 mentions)
Abc system, by Vector Laboratories (2 mentions)
Biotinylated goat anti rat secondary antibody, by Jackson ImmunoResearch (1 mentions)
Biotinylated goat anti rat, by Vector Laboratories (1 mentions)
Nanozoomer digital pathology system, by Hamamatsu Photonics (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Mbp antibody, by Abcam (1 mentions)
Phase contrast light microscope, by Nikon (1 mentions)
Ab52599, by Abcam (1 mentions)
Sc 73632, by Santa Cruz Biotechnology (1 mentions)
Tgfbr1, by Abcam (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Snail1, by Cell Signaling Technology (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab2302, by Abcam (1 mentions)
8 protocols using diaminobenzidine dab kit
1
BrdU Immunohistochemistry Protocol
2
BrdU Immunohistochemistry for Dividing Cells
3
Microglial Morphology Analysis in Piglet Hippocampus
4
Western Blot Analysis of Corpus Callosum Proteins
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Protein extract from corpus callosum were centrifuged at 560 g for 5 min; then stored at -20°C. To ensure loading of equal amounts during Western blotting, protein concentration of each sample was determined with Bradford assay. Equivalent amounts of total protein extract from each sample were mixed with sample buffer, boiled, and loaded onto SDS polyacrylamide gels. Electrophoretic separation of the extracts was typically performed on 7.5–15% gel. The proteins were then transferred to polyvinylidene (PVDF) (Gibco, CA) and probed with MBP antibody (1:3000 in blocking buffer) (Abcam, USA) and PLP antibody (1:1000 in blocking buffer) (Abcam, USA) for an overnight on shaker in 4°C. Membranes were subsequently rinsed in blocking buffer (4 times/3 min each time) and stained with secondary antibody (1:1000 in blocking buffer) (Abcam, USA) for 1 hour. Then, membranes were washed with TBS Tween-20 blocking buffer (3 times) and the proteins were visualized with diaminobenzidine (DAB) kit (Sigma, USA). The membranes were scanned and the photos were analyzed with image 2 software.
5
Immunohistochemical Analysis of H2B
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All tissues were fixed in a formalin solution, dehydrated in ethanol, embedded in paraffin, and sectioned. After blocking, the slides were incubated with anti-H2B (Abcam, ab52599) overnight at 4°C. After washing with PBS, the slides were incubated with goat anti-rabbit horseradish peroxidase (HRP) (Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. A diaminobenzidine (DAB) kit (Sigma-Aldrich, St. Louis, MO, USA) was used to detect the immunohistochemical reactions. The slides were examined under a phase contrast light microscope (Nikon). Three areas were chosen randomly from each section for measurement.
6
Histological and Immunohistochemical Analysis of Dental Pulp Tissue Regeneration
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The transplants were extracted 8 weeks after operation, fixed with 10% formaldehyde solution, decalcified with ethylene diamine tetraacetic acid (EDTA), and embedded in paraffin. A series of 5 μm sections were cut, and the sections were stained with hematoxylin-eosin (HE) for histological analysis. Immunohistochemistry was performed to analyze the newly formed tissue. The sections were incubated with primary antibodies against DSPP (sc-73632, 1:100, Santa Cruz Biotechnology Inc.), GFP (1:100, Abcam), and CD31 (1:100, Abcam) overnight at 4°C. The slides were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody, a diaminobenzidine (DAB) kit (Sigma) was used to stain the slides, and the sections were counterstained with hematoxylin. For DSPP staining, mouse femur bone tissue was treated as negative control. The number of blood vessels within the newly formed dental-pulp-like tissue was calculated using the average value of the three parallel slices (40× magnification) selected from each of the three equally divided paraffin parts along the cross-section.
7
Immunohistochemical Analysis of Xenograft Tumors
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Xenograft tumor specimens from nude mice were fixed in 4% paraformaldehyde and subsequently embedded in paraffin. After dewaxing and hydration, antigen retrieval and blocking, the 5μm sections were incubated with specific primary antibodies overnight at 4°C. Primary antibodies were as follows: TGFBR1(1:200, Abcam, MA, USA), E-cadherin (1:300, Cell Signaling Technology, MA, USA), Vimentin (1:200, Cell Signaling Technology, MA, USA) and Snail1(1:200, Cell Signaling Technology, MA, USA). Then the sections were incubated with anti-mouse/ rabbit secondary antibody (Abcam, USA) for 30min at room temperature. Staining was performed using diaminobenzidine (DAB) kit (Sigma, USA). The images were obtained under a microscope (Olympus, Japan) with appropriate magnification.
8
Immunohistochemical Analysis of Xenograft Tumors
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Xenografted tumor specimens from nude mice were fixed in 4% paraformaldehyde and then embedded in paraffin. After dewaxing, hydration, antigen retrieval, and sealing, 5 μm sections were incubated overnight with specific primary antibodies at 4°C. Primary antibodies were as follows: Ki67 (ab15580, Abcam, 1: 1000), cleaved caspase-3 (ab2302, Abcam, 1: 1000), Bax (ab32503, Abcam, 1: 1000), Bcl-2 (12789-1-AP, Proteintech, 1: 1000). The sections were then incubated at room temperature for 30 min with anti-mouse/rabbit secondary antibody (Abcam, USA). Diaminobenzidine (DAB) kit (Sigma, USA) was used for staining. Images were obtained under a microscope with appropriate magnification (Olympus, Japan).
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