Frozen sections of human tonsil, normal, and asthma lung tissue (Biochain) were blocked with 10% goat serum and then stained with anti-SEMA4A mAb (house hold, 1:200 dilution) at room temperature for 2 h. After washing, Horseradish Peroxidase (HRP)-conjugated goat anti-mouse antibody (ab19194, 1:2000 dilution) from Abcam was applied to stain for 15 min. The slides were washed and incubated with substrate DAB Chromogen (D7304, Sigma). Finally, Haematoxylin (H3136, Sigma) was applied to counter stain according to the manufacture’s instruction. For double immunofluorescence staining, the slides were stained with anti-SEMA4A mAb, followed by Alexa Fluor 594-labeled anti-mIgG antibody (A-11020, 1:500 dilution) from Thermo Fisher Scientific. Meanwhile, anti-CD11c Ab (NB110-40766, 1;200 dilution) from Novus Biologicals and Alexa Fluor 488-labeled Goat Anti-Rabbit IgG H&L Abs (ab150077, 1:500 dilution) from Abcam were applied for staining. Images were acquired by using an inverted microscope, BX41 (Olympus, Tokyo).
Dab chromogen
Manufactured by Merck Group
Sourced in United States, United Kingdom
DAB chromogen is a laboratory reagent used in immunohistochemistry (IHC) and related techniques. It serves as a substrate for the enzyme-mediated detection of target molecules in biological samples. DAB chromogen produces a brown-colored precipitate at the site of the target, allowing for visualization and identification of the labeled structures.
Automatically generated - may contain errors
Lab products found in correlation
Target retrieval solution, by Agilent Technologies (3 mentions)
Mayer s hematoxylin, by Agilent Technologies (2 mentions)
Dual endogenous enzyme blocking reagent, by Agilent Technologies (2 mentions)
Powervision poly hrp ihc detection system protocol, by Leica (2 mentions)
Haematoxylin, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse antibody, by Abcam (1 mentions)
Vector abc kit, by Vector Laboratories (1 mentions)
Multi gauge software, by Fujifilm (1 mentions)
Goat serum, by Abcam (1 mentions)
Rabbit anti myelin basic protein mbp, by Abcam (1 mentions)
Dual endogenous enzyme block, by Agilent Technologies (1 mentions)
Powervision kit, by Leica (1 mentions)
P7949, by Merck Group (1 mentions)
Mcap497, by Bio-Rad (1 mentions)
8 protocols using dab chromogen
1
Immunohistochemical Detection of SEMA4A
2
Immunohistochemical Detection of SYT1 in Postmortem Brain
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To detect SYT1 in human postmortem brain tissues, we performed immunohistochemistry as described previously55 (link). Coronal plane of paraffin-embedded tissue sections (10 μm) were incubated with blocking solution after 3% H2O2 reaction for 1 hr. The tissue sections were incubated with anti-synaptotagmin 1 antibody (1:100 dilution; Abcam, ab131551) for 24 hr. After secondary antibody reaction, the tissue slides were further processed with Vector ABC Kit (Vector Lab PK-6100). DAB chromogen (Sigma D5637) was used to develop the immunoreactive signals. The nuclei were counterstained with hematoxylin. The tissue slides were examined under a bright field microscope and the intensity of immunoreactivity was analyzed using Multi-Gauge Software (Fuji photo film Co, Ltd. Japan).
3
Immunohistochemical Staining of ADRM1 in FFPE Tissues
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RPN13 was stained by following PowerVision Poly-HRP IHC Detection system protocol (Leica Biosystem). FFPE sections were deparaffinized in xylene, followed by rehydration in graded ethanol. Antigen retrieval was performed by steaming specimens at 100 °C for 20 min in Target Retrieval Solution (Dako) and subsequently washed in Tris-buffered saline with Tween 20 (TBST, 0.05% Tween 20). Endogenous peroxidase was blocked, by treatment of slides with Dual Endogenous Enzyme-Blocking Reagent (Dako) for 5 min at room temperature. Sections were covered with 1:500 dilution of mouse monoclonal ADRM1 Antibody (F-12) supplied by Santa Cruz Biotechnology (sc-166,754) diluted with Antibody Dilution Buffer (ChemMate) and then incubated at room temperature for 45 min. Slides were then washed with TBST, followed by incubation with PowerVision Poly-HRP Anti-Mouse IgG for 30 min at room temperature. After three washes in TBST, sections were treated with DAB chromogen (3, 3 ‘-diaminobenzidine tetrahydrochloride; Sigma) for 20 min in the dark. Sections were counterstained with Mayer’s hematoxylin (Dako), dehydrated with ethanol and xylene, and mounted permanently.
4
Immunohistochemical Staining of CD3 in FFPE Tissue
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CD3 was stained by following PowerVision Poly-HRP IHC Detection system protocol (Leica Biosystem). Briefly, FFPE sections were deparaffinized in xylene, followed by dehydration in graded ethanol. Antigen retrieval was performed by steaming specimens at 100°C for 20 min in Target Retrieval Solution (Dako) and subsequently washed in Tris-buffered saline with Tween 20 (TBST, 0.05% Tween 20). Endogenous peroxidase was blocked, by treatment of slides with Dual Endogenous Enzyme-Blocking Reagent (Dako) for 5 min at room temperature. Sections were covered with rabbit monoclonal CD3 primary antibody (ThermoFisher, RM-9107, 1:300) diluted with Antibody Dilution Buffer (ChemMate) and then incubated at room temperature for 45 min. Slides were then washed with TBST, followed by incubation with PowerVision Poly-HRP Anti-Rabbit IgG for 30 min at room temperature. After three washes in TBST, sections were treated with DAB chromogen (3, 3 '-diaminobenzidine tetrahydrochloride; Sigma) for 20 min in the dark. Sections were counterstained with Mayer’s hematoxylin (Dako), dehydrated with ethanol and xylene, and mounted permanently.
5
Immunohistochemical Staining of Myelinated Fibers
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Perfused brains were cut into 30 µm sections coronally using the Leica Vibrating Microtome (model VT1200; Leica Microsystems, Inc.). Floating sections were quenched with 0.3% hydrogen peroxide in 0.01 M PBS at room temperature (RT) for 30 min to remove endogenous peroxidase activity. The sections were subsequently incubated in 0.01 M PBS with 10% goat serum (Abcam) blocking solution for 1 h at RT and then incubated overnight with rabbit anti-myelin basic protein (MBP; 1:250; cat. no. 78896; Cell Signaling Technology, Inc.) primary antibody diluted in the blocking solution. After washing in 0.01 M PBS, the sections were incubated with goat anti-rabbit IgG biotin-conjugated secondary antibody (1:1,000; cat. no. BA-1000-1.5; Vector Laboratories, Inc.) for 1 h at RT. The Avidin-Biotin Complex Kit (cat. no. PK-6100; Vector Laboratories, Inc.) was used according to manufacturer's instructions and visualized by incubating for 1-2 min at room temperature in DAB chromogen (Sigma-Aldrich; Merck KGaA), monitoring for color development.
6
Immunohistochemical Analysis of sCJD Prion Deposits
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Sections of paraformaldehyde-fixed, paraffin-embedded human cerebellar tissue samples from a patient with diagnosed sporadic CJD (sCJD) with primitive plaques and synaptic prion deposition pattern were used in the study. Tissue samples were immersed in 96% formic acid for 1 h after fixing in paraformaldehyde. Sections were deparaffinized and pretreated for optimal antigen retrieval by 30 min autoclaving at 121 °C in distilled water, followed by a 5 min incubation in 96% formic acid. The sections were then blocked in 1% BSA solution for 20 min at RT. They were subsequently incubated overnight at RT in a moist chamber with all N-terminal mAbs tested at the concentration of 5 µg/mL. All sections were then washed and incubated for 1.5 h with anti-mouse HRP-labeled antibodies diluted 1:1000 (Jackson ImmunoResearch, West Grove, Pennsylvania, USA) at RT. After thorough rinsing, the sections were developed in DAB chromogen (Sigma, St. Louis, Missouri, USA) for 5 min. Brain tissue counterstaining was obtained by immersion of sections in Mayer’s hematoxylin for 2 min.
7
Immunostaining of Lung Sections
8
Immunohistochemical Analysis of Caspase-3 Expression
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Immunohistochemical staining is done using the technique of paraffin block fixation of neutral buffered formalin. Caspase-3 polyclonal antibody rabbit polyclonal anti-mouse caspase-3 (Dako Glostrup, Denmark) is used as primary antibody and anti-rabbit biotin-conjugated is used as secondary antibody (Dako Glostrup, Denmark). The expression of caspase-3 was seen microscopically by observing brownish staining because the anti-caspase-3 antibody reaction is visualized by DAB chromogen (Sigma Aldrich, UK). The observation was performed using a light microscope (Olympus) at 10 visual fields with 400 times magnification. Caspase-3 expression was calculated in the cytoplasm of colon epithelial cells that showed a brownish color.
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