3‐μm sections from human core samples or mouse lung were deparaffinized, rehydrated, and then treated with 1.8% (v/v) H2O2 solution (Sigma‐Aldrich) to block endogenous peroxidase. Heat‐induced epitope retrieval was performed in HIER citrate buffer (pH 6.0, Zytomed Systems) in a decloaking chamber (Biocare Medical). To inhibit non‐specific binding of antibodies, sections were treated with a blocking antibody (Biocare Medical). Human sections were incubated at 4°C overnight with a rabbit anti‐TLR4 primary antibody (1:50, Cat. No. ab13556, Abcam), followed by 1 h with an anti‐rabbit HRP‐conjugated secondary antibody (Biocare Medical). Signals were amplified by adding chromogen substrate 3,3′‐diaminobenzidine (DAB; Biocare Medical). Mouse sections were incubated at 4°C overnight with a rabbit anti‐galectin‐3 primary antibody (1:100, Cat. No. sc‐20157, Santa Cruz Biotechnology), followed by 1 h with a Rabbit‐on‐Rodent AP‐Polymer (Biocare Medical). Signals were amplified by adding chromogen substrate Vulcan fast red (Biocare Medical). All sections were counterstained with hematoxylin (Sigma‐Aldrich), dehydrated, and mounted.
3 3 diaminobenzidine dab
Manufactured by Biocare Medical
Sourced in United States
3,3′-diaminobenzidine (DAB) is a common chromogenic substrate used in immunohistochemistry and other biological applications. It is used to visualize the presence and localization of target proteins or molecules in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin, by Merck Group (2 mentions)
Goat anti mouse nkp46 ncr1, by R&D Systems (2 mentions)
Goat on mouse hrp polymer kit ghp516, by Biocare Medical (2 mentions)
Eukitt, by Merck Group (2 mentions)
Cell f software, by Olympus (2 mentions)
Hier citrate buffer, by Zytomed Systems (1 mentions)
Anti rabbit hrp conjugated secondary antibody, by Biocare Medical (1 mentions)
Decloaking chamber, by Biocare Medical (1 mentions)
Vulcan fast red, by Biocare Medical (1 mentions)
H2o2 solution, by Merck Group (1 mentions)
Silanized slides, by Agilent Technologies (1 mentions)
Ds ri2, by Nikon (1 mentions)
Rat anti mouse ly6g, by BD (1 mentions)
Goat on rodent, by Biocare Medical (1 mentions)
Goat anti mouse cd31, by R&D Systems (1 mentions)
Mach 1, by Biocare Medical (1 mentions)
Rat anti mouse cd3, by Bio-Rad (1 mentions)
Rat on mouse hrp polymer kit, by Biocare Medical (1 mentions)
Rabbit anti mouse iba1, by Fujifilm (1 mentions)
Buffered formalin, by Thermo Fisher Scientific (1 mentions)
13 protocols using 3 3 diaminobenzidine dab
1
Immunohistochemical Detection of TLR4 and Galectin-3
2
Immunohistochemical Analysis of Stomach Sections
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Stomach sections (5 μm thick) from 5 animals per group were obtained using a microtome and then transferred onto silanized slides (Dako, Glostrup, Hovedstaden, Denmark). These sections underwent dewaxing and hydration procedures. Subsequently, a simple indirect blocking method was employed using Anti Peroxidase Peroxidase (APP), wherein the primary antibody targeted the antigen (protein) to be detected, while the secondary antibody facilitated binding to the APP complex. The slides were then incubated overnight at 4 °C with primary antibodies against NF-kB and TGF-β. Following thorough washing with distilled water, the slides were treated with a secondary antibody for 60 min. 3,3′-diaminobenzidine (DAB, Biocare Medical, Pacheco, CA, USA) was utilized as the chromogen, and the specimens were counterstained with hematoxylin. The samples were examined under an optical microscope (Olympus microscope, Tokyo, Japan) equipped with a camera (Nikon DS-Ri2).
3
Comprehensive Necropsy and Histopathological Analysis
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A full necropsy was performed, and gross observations were recorded in all mice. The following organs were collected: adrenal glands, BM (right femur and tibia, sternum), brain, cecum, colon, duodenum, eyes with optic nerves, heart, ileum, jejunum, kidneys, liver together with gall bladder, lungs, lymph nodes (mandibular and mesenteric), rectum, salivary glands, spleen, stomach, testes, thymus, and macroscopic abnormalities. Organs were fixed in 10% buffered formalin, trimmed and embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin for histological examination. Selected slides were stained with monoclonal rat anti-human CD3 (Bio-Rad), rat anti-mouse B220/CD45R (clone RA3-6B2; Bio-Rad), and rat anti-mouse F4/80 (clone CI: A31; Bio-Rad) after antigen retrieval. Immunoreactions were revealed using a rat on rodent HRP-polymer (Biocare Medical) and the 3,3 diaminobenzidine (DAB) as chromogen (Biocare Medical). Slides were then counterstained with hematoxylin. Histopathological examination was performed by board-certified pathologists on all tissues of the study animals. A peer review was performed by a second board-certified pathologist on all mice with significant histological findings, including all neoplasia, according to GLP SR-TIGET Test Facility Standard Operating Procedures.
4
Immunohistochemical Staining of Murine Tissues
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Paraffin-embedded murine tissues were cut at 3 um and placed on super-frost slides. After dewaxing and rehydration, antigen unmasking was performed with Decloaking Chamber in DIVA Buffer 1X (DV2005L2J Biocare Medical, Pacheco, CA, USA) (3 min at 125 °C, 5 min at 90 °C) (CD31, CD3, Ly6G); for IBA1 staining, antigen unmasking was not performed. Endogenous peroxidases were blocked with 2% H2O2 for 20 min and then rodent block M (for IBA1) or PBS/BSA (bovin serum albumin) 2% (for CD31, CD3, and Ly6G staining) were used to block unspecific binding sites. Sections were incubated with the following antibodies: rabbit anti-mouse IBA-1 (1:250, Wako), goat anti-mouse CD31 (1:1000, R&D), rat anti-mouse CD3 (1:1000, Serotec), and rat anti-mouse Ly6G (1:200, BD Biosciences). All the primary antibodies were incubated for 1 h in a humid chamber at room temperature. As secondary antibody, we used a Rat on Mouse HRP polymer kit (Biocare Medical) (CD3, Ly6G), Goat on Rodent (Biocare Medical) (CD31), and Mach1 (Biocare Medical) (IBA1). Reactions were developed with 3,3′-diaminobenzidine, DAB (Biocare Medical) and then counterstained with hematoxylin and mounted with Eukitt.
5
Immunohistochemical Analysis of Breast Tumor Tissues
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Tumor tissues collected from Z-endoxifen and tamoxifen-randomized and letrozole-treated MCF7LR tumors were fixed overnight in buffered formalin (Fisher Scientific) and processed in the tissue core facility at Mayo Clinic (Scottsdale, AZ). Deparaffinized and rehydrated 5- to 6-μm sections were unmasked for 15 min in Citrate Buffer (pH 6.0) at 95 to 99 °C. Primary antibodies against phospho-Akt (Ser473) (CS#4060) at 1:100 dilution were incubated overnight at 4 °C. Secondary antibody (CS #8114) was applied for 30 to 60 min at room temperature. For Ki-67 staining of the tumoral tissues, primary antibodies against Ki-67 (Clone MIB-1) (Dako North America) at 1:600 were incubated overnight at 4 °C. Secondary antibody (Cell Signaling; SignalStain Boost IHC detection system #8125S) was applied for 30 to 60 min at room temperature. Chromogenic detection of protein expression was determined in the presence of 3,3′-diaminobenzidine (DAB) (BioCare) and visualized by light microscopy. Ki-67 was quantitated as percentage of tumor nuclei with staining.
6
Quantifying TGF-β1 Expression in Tracheal Scars
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In situ TGF-β1 expression in the tracheal scar was assessed by immunohistochemistry (IHC) of the tracheal tissue sections used for microscopy with a rabbit polyclonal anti-TGF-β1 antibody (Abcam ab25121, USA) using the biotin-streptavidin-peroxidase system (Vectastain Universal Quick Kit, Burlingame, CA). The sections were incubated with 3,3′-diaminobenzidine (DAB, BioCare Medical, USA) and counterstained with CAT hematoxylin (BioCare Medical, USA). After completing the staining, TGF-β1 expression was quantified in the entire sample circumference using the program ImageJ (http://rsbweb.nih.gov/ij/ ), which was developed by the National Institutes of Health (NIH), and the plugin IHC Profiler [17 (link)].
7
Immunohistochemical Analysis of NKp46 in Liver Tissues
8
Immunohistochemical Analysis of NKp46 in Liver Tissues
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Liver frozen tissues were cut at 8 mm and then fixed with 4% PFA. Endogenous peroxidases were blocked with 0.03% of H2O2 for 5 min and unspecific binding sites were blocked with PBS + 1% FBS for 1h. Tissues were stained with polyclonal goat anti mouse NKp46/NCR1 (R&D System) and goat on mouse HRP polymer kit (GHP516, Biocare Medical) was used as secondary antibody. Reactions were developed with 3,3’-Diaminobenzidine (DAB) (Biocare Medical) and then slides were counterstained with hematoxylin. Slides were mounted with eukitt (Sigma-Aldrich). 20X images were analyzed with cell^F software (Olympus).
9
Immunocytochemistry Confirms MT-2 Activity
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An immunocytochemistry method was used to confirm the TTSPs activity results obtained by the above-mentioned method. For the immunostaining, we used 105 normal and iron deficiency HepG2 cells model, similar to the MT-2 activity study design, which were seeded into a 24-well plate with coverslips for 24 h. The following day, all HepG2 cells were incubated with 1:200 diluted monoclonal antibody anti-human MT-2 (Santa Cruz Biotechnology, US) for overnight. To detect MT-2 expression in the HepG2 cells, a secondary antibody conjugated streptavidin–horseradish peroxidase (Biocare, US) was used to generate antigen–antibody complexes. 3,3′-Diaminobenzidine (DAB) (Biocare, US) and Hematoxylin-Eosin (Merck, US) staining were used to visualize the presence of MT-2 protein in the HepG2 cells under a light microscope. Manual calculation of MT-2 expression was conducted, in a minimum of five fields, by two independent pathologists using the Image Raster software (https://www.miconos.co.id/p/download.html ). The MT-2 expression is presented as the percentage of immunopositive cells divided by all iron deficiency HepG2 cells.
10
Caspase-3 Immunohistochemistry in Heart and Brain
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Immunohistochemical examination was applied for a sensitive marker for apoptosis (caspase-3) following the manufacturer’s instructions (Biocare Medical, Pacheco, CA, USA). The paraffin was segregated from the paraffin-embedded third portion of the heart and brain sections before the endogenous peroxidase activity was frustrated with 3% H2O2. Citrate buffer used in antigen retrieval achievement. Then, 5% BSA was conducted on the heart and brain sections to remove any nonspecifically bound protein after cooling. Then, the sections were brooded overnight (4 °C) with primary antibody (rabbit monoclonal anticaspase-3). PBS cleaned the sections, afterward; they were incubated with a secondary conjugated polymer for 30 min at RT on the next day. The next step was incubation for 5 min RT with Biocare’s 3,3-diaminobenzidine (DAB) or for 6–8 min at 25 °C with Biocare’s Warp Red Counterstain with Mayer’s hematoxylin, followed by soaking with deionized water. Tacha’s Bluing Solution was performed for 1 min, followed by wetting with deionized water. Biocare’s MACH 4 detection system was employed for standardizing this antibody. Pictures of sections were taken under a microscope (Nikon) (Gown and Willingham 2002 ).
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