Frozen brain material from active MS lesions was obtained from The Netherlands Brain Bank. Clinical patient details are depicted in Additional file 1 : Table S1. Following acetone fixation (10 min) and blocking (30 min), serial cryosections were stained overnight for proteolipid protein (PLP) to assess demyelination and CD68 or HLA-DR to assess the presence and distribution of microglia/macrophages [30 (link)]. Staining was visualized with HRP-labelled secondary antibodies (Envision+, Dako) and 3,3′-Diaminobenzidine (DAB) substrate-chromogen (Dako). Similarly, sections were stained with antibodies against protein-bound acrolein (1/100, mouse monoclonal, 10A10, Novus Biologicals). For fluorescent double-staining, acrolein-protein adducts were visualized with secondary goat anti-mouse (1/600, AF 555, A-21425, Thermo Fisher), followed by overnight incubation with primary antibodies against CD68 (1/100, mouse monoclonal, M0814 KP1 clone, Dako) or glial fibrillary acidic protein (GFAP, 1/100, mouse monoclonal, G3893, Sigma), and secondary goat anti-mouse (1/400, IgG1 AF 488, A-21121, Thermo Fisher). Imaging was performed with a Leica DM4000 B LED (Leica Microsystems).
3 3 diaminobenzidine dab substrate chromogen
Manufactured by Agilent Technologies
Sourced in Denmark, United States
3,3'-diaminobenzidine (DAB) substrate-chromogen is a laboratory reagent used in immunohistochemistry and other analytical techniques. It serves as a chromogenic substrate that produces a brown precipitate when catalyzed by peroxidase enzymes, enabling the visualization and detection of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Leica dm4000 b led, by Leica Microsystems (1 mentions)
Af555, by Thermo Fisher Scientific (1 mentions)
A21425, by Thermo Fisher Scientific (1 mentions)
G3893, by Merck Group (1 mentions)
Envision, by Agilent Technologies (1 mentions)
Antibody diluent, by Agilent Technologies (1 mentions)
Ab53444, by Abcam (1 mentions)
Alexa fluor 488 donkey anti rat, by Thermo Fisher Scientific (1 mentions)
Af1152, by R&D Systems (1 mentions)
Donkey anti rat hrp, by Jackson ImmunoResearch (1 mentions)
α sma clone 1a4, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
F4 80, by Cell Signaling Technology (1 mentions)
Envision complex, by Agilent Technologies (1 mentions)
Foxp3, by Cell Signaling Technology (1 mentions)
Aperio cs2, by Leica Biosystems (1 mentions)
Envision flex target retrieval solution, by Agilent Technologies (1 mentions)
Flex ihc microscope slides, by Agilent Technologies (1 mentions)
Labelled polymer hrp, by Agilent Technologies (1 mentions)
5 protocols using 3 3 diaminobenzidine dab substrate chromogen
1
Histochemical Analysis of Active MS Lesions
2
Immunohistochemical Analysis of RCC Tumors
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The construction of the tissue microarray consisting of 453 RCC tumors as well as the immunohistochemical analyses and applied antibodies have been already reported in more detail in Jasinski-Bergner and co-authors, 2015 [8 (link)]. Immunohistochemistry (IHC) of 5 μm sections was performed by using the HLA-G-specific mAb [4H84] (Abcam, Cambridge, UK) at a 1:50 dilution generated with the Antibody Diluent (Dako, Hamburg, Germany). As secondary antibody a HRP-linked anti-mouse and the 3,3′-diaminobenzidine (DAB+) substrate chromogen (Dako) was employed.
The evaluation and the pathological diagnostics were performed by the pathologists Prof. A. Hartmann and Dr. V. Spath. The generation of the tissue microarray was performed according the principles expressed in the declaration of Helsinki.
The evaluation and the pathological diagnostics were performed by the pathologists Prof. A. Hartmann and Dr. V. Spath. The generation of the tissue microarray was performed according the principles expressed in the declaration of Helsinki.
3
Quantifying Cellular Markers in Tissue Sections
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Primary antibodies were anti–Gal-1 (AF1152, R&D Systems), CD68 (Ab53444, Abcam), and α-SMA (clone 1A4, Sigma-Aldrich). Rabbit anti-goat horseradish peroxidase (HRP), donkey anti-rat HRP (The Jackson Laboratory), and Alexa Fluor 488 donkey anti-rat (Invitrogen) were used as secondary antibodies. HRP was then added, and sections were stained with 3,3′-diaminobenzidine (DAB) substrate-chromogen (DAKO) and counterstained with hematoxylin. Computer-assisted morphometric analysis was performed with Image-Pro Plus software (version 1.0 for Windows). The threshold setting for area measurement was equal for all images. Samples from each animal were examined in a blinded manner. Results were expressed as % positive area versus total area (CD68 and α-SMA).
4
Immunohistochemical Analysis of Mouse Tissues
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Organs were fixed for 24 h in 3.7%–4% w/v formaldehyde buffered to pH 7 and shaken at room temperature, followed by incubation for another 48 h in 70% ethanol. Tibias were decalcified in 4% ethylenediaminetetraacetic acid (EDTA) before embedding in paraffin.
Paraffin-embedded sections of mouse tibias and lungs were immunostained with antibodies specific to Ki67 (1:500; Cell Signaling Technology; 12202S), caspase-3 (1:500; Cell Signaling Technology; 9662), CD3 (1:150; Abcam; ab16669), CD8 (1:500; Cell Signaling Technology; 98941), Foxp3 (1:100; Cell Signaling Technology; 12653), CD31 (1:100; Cell Signaling Technology; 77699), GR1 (1:3,000; BioLegend; 108402), and F4/80 (1:200; Cell Signaling Technology; 70076), following conventional procedures and according to the manufacturers’ instructions. Immunodetection was performed using biotinylated secondary antibodies and streptavidin horseradish peroxidase (HRP) complex (EnVision complex; Dako), and immune complexes were visualized with 3,3′-diaminobenzidine (DAB) substrate-chromogen (Dako, Glostrup, Denmark), followed by counterstaining with hematoxylin. Slides were scanned at 20× using Aperio CS2 (Leica Biosystem, Barcelona, Spain), regions of the samples were extracted using Aperio ImageScope version (v.)12.3.2.8013, and quantification was performed using Fiji/ImageJ platform 18.
Paraffin-embedded sections of mouse tibias and lungs were immunostained with antibodies specific to Ki67 (1:500; Cell Signaling Technology; 12202S), caspase-3 (1:500; Cell Signaling Technology; 9662), CD3 (1:150; Abcam; ab16669), CD8 (1:500; Cell Signaling Technology; 98941), Foxp3 (1:100; Cell Signaling Technology; 12653), CD31 (1:100; Cell Signaling Technology; 77699), GR1 (1:3,000; BioLegend; 108402), and F4/80 (1:200; Cell Signaling Technology; 70076), following conventional procedures and according to the manufacturers’ instructions. Immunodetection was performed using biotinylated secondary antibodies and streptavidin horseradish peroxidase (HRP) complex (EnVision complex; Dako), and immune complexes were visualized with 3,3′-diaminobenzidine (DAB) substrate-chromogen (Dako, Glostrup, Denmark), followed by counterstaining with hematoxylin. Slides were scanned at 20× using Aperio CS2 (Leica Biosystem, Barcelona, Spain), regions of the samples were extracted using Aperio ImageScope version (v.)12.3.2.8013, and quantification was performed using Fiji/ImageJ platform 18.
5
Immunohistochemistry for H. pylori Detection
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For H. pylori detection, immunohistochemistry was performed on 4 µm paraffin sections and mounted on FLEX IHC Microscope Slides (Dako, United States). Briefly,
sections were deparaffinized and re hydrated followed by heat-induced epitope retrieval using EnVision FLEX Target Retrieval Solution (Dako, United States). After
blocking endogenous peroxidase with a 2% H2O2 solution, sections were incubated with the primary polyclonal rabbit anti- H. pylori antibody (Dako, United States) with
a dilution of 1:10 at room temperature for 30 minutes. After sections were washed, a labelled-polymer- HRP (Dako, United States) was applied for 20 minutes. Finally,
sections were incubated with 3, 3'-diaminobenzidine (DAB)+ substrate-chromogen (Dako, United States) for 10 min, and washed in tap water. Then, slides were immersed with
hemato xylin and mounted. As a positive control, a gastric mucosal tissue infected with H. pylori was taken.
sections were deparaffinized and re hydrated followed by heat-induced epitope retrieval using EnVision FLEX Target Retrieval Solution (Dako, United States). After
blocking endogenous peroxidase with a 2% H2O2 solution, sections were incubated with the primary polyclonal rabbit anti- H. pylori antibody (Dako, United States) with
a dilution of 1:10 at room temperature for 30 minutes. After sections were washed, a labelled-polymer- HRP (Dako, United States) was applied for 20 minutes. Finally,
sections were incubated with 3, 3'-diaminobenzidine (DAB)+ substrate-chromogen (Dako, United States) for 10 min, and washed in tap water. Then, slides were immersed with
hemato xylin and mounted. As a positive control, a gastric mucosal tissue infected with H. pylori was taken.
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