Ten HL patient samples and 5 ALCL patient samples were obtained at the time of relapse or progressive disease and analyzed by IHC. Samples were obtained from leftover tissue on COH IRB approved protocol. For CD30 immunostaining, we used the monoclonal mouse anti-human CD30, clone Ber-H2. For MDR1 immunostaining, we used the monoclonal mouse anti-human MDR1, clone PG-M1. Both immunostaining was visualized with DAKO Envision/HRP kit (Dakocytomation). Immunohistochemical staining was performed on 5-μm thick paraffin embedded tissue. Tissue sections were deparaffinized in xylene followed by 100%–70% alcohol. Samples were then quenched in 3% hydrogen peroxide and pretreated to promote antigen retrieval with High pH. Slides were incubated in primary antibody at 1/30 dilution for 20 minutes at RT. After rinsing in Dako Wash, slides were incubated in EnVision FLEX/HRP (DAKO K-8000) for 20 minutes. Slides were further washed in Dako buffer and then incubated with diaminobenzidine tetrahydrochloride (DAB) from Dako, counterstained with hematoxylin, and mounted.
Diaminobenzidine tetrahydrochloride dab
Manufactured by Agilent Technologies
Sourced in Denmark
Diaminobenzidine tetrahydrochloride (DAB) is a chromogenic substrate used in immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) to detect the presence of specific proteins or antigens. It produces a brown precipitate at the site of the enzyme-catalyzed reaction, allowing for visualization and detection of the target molecule.
Automatically generated - may contain errors
Lab products found in correlation
Dako envision kit hrp, by Agilent Technologies (1 mentions)
Envision flex hrp, by Agilent Technologies (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Bovine serum albumin (bsa), by Agilent Technologies (1 mentions)
Mab1433, by R&D Systems (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Rabbit anti human cd3, by Agilent Technologies (1 mentions)
Horseradish peroxidase conjugated anti mouse antibody, by Agilent Technologies (1 mentions)
Ipp version 6, by Media Cybernetics (1 mentions)
Confocal microscopy, by Leica (1 mentions)
Live dead double staining solution, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti mouse secondary antibody, by Agilent Technologies (1 mentions)
6 protocols using diaminobenzidine tetrahydrochloride dab
1
Immunohistochemical Analysis of CD30 and MDR1 in Relapsed ALCL
2
Quantification of Tumor-Infiltrating T Cells
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Paraffin-embedded surgical specimens of both the primary tumor and corresponding liver metastasis of the same patients were collected after surgical resection. In patients with multiple liver lesions, a random metastatic specimen was selected to represent the immune status of all metastases. All of the specimens were sectioned and stained with immunohistochemical techniques that labeled the CD3+ and CD8+ T cells with specific antibodies: a rabbit anti-CD3 monoclonal antibody (ZSGS-BIO; Beijing, China; catalogue number: ZM-0417; dilution: commercial working solution) and a rabbit anti-CD8 monoclonal antibody (ZSGS-BIO; catalog number: ZA-0508; dilution: commercial working solution). After incubation with primary antibodies at 4 °C overnight, the slides were treated with a detection reagent mixture that included the corresponding secondary antibody, a streptavidin-horseradish peroxidase complex and diaminobenzidine tetrahydrochloride (DAB) (Dako, Santa Clara, CA, USA; catalog number: K5007) and were incubated in the dark at 37 °C for 30 minutes. Images of each slide were captured by a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan) at 20× magnification.
3
Immunohistochemical Quantification of Osteopontin
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After dewaxing in three toluene baths of 3 minutes each, the sections were rehydrated by passage through decreasing gradients of alcohol baths. After that, the sections were subjected to antigen unmasking by heat in a citrate buffer at pH 6 for 10 minutes. Then, 0.1% sodium azide (Sigma‐Aldrich) was used to inhibit endogenous peroxidases in the presence of 0.3% hydrogen peroxide for 30 minutes. After washing with PBS (Invitrogen), 10% BSA (Dako) was added for a period of 30 minutes. Then, the sections were incubated with anti‐OPN polyclonal primary antibodies (MAb1433, 1:1000, R&D Systems). Isotype antibodies were used as negative controls. After rinsing, a biotinylated rabbit anti‐rat secondary antibody (Dako) was added for 30 minutes; then, a biotinylated streptavidin‐peroxidase complex (Dako) is applied for 30 minutes. Diaminobenzidine tetrahydrochloride (DAB) (Dako) was used for development. Finally, Haematoxylin‐Meyer was used for counterstaining. After image acquisition, ImageJ software was used for quantification.
4
Quantifying Immune Cell Types in Skin Tissue
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Tissue sections of the paraffin-embedded biopsies were stained with haematoxylin–eosin (HE) for the detection of eosinophils, toluidine blue staining for mast cells and two immunohistochemical stainings (CD3 staining for T-cells and CD20 staining for B-cells), based on Dreesen et al. [31 (link)]. In short, skin tissue sections of 4 μm were mounted on APES-coated slides, blocked with H2O2 and stained with polyclonal rabbit anti-human CD3 (Dako, Belgium) or rabbit anti-human CD20 (Sigma-Aldrich, USA) antibodies. T- and B-cells were visualized by adding peroxidase labelled goat anti-rabbit antibodies (Dako, Belgium), diaminobenzidine tetrahydrochloride (DAB; Dako, Belgium) and by performing a counterstaining with haematoxylin. Eosinophils, mast cells, T-cells and B-cells were quantified by taking two random pictures per tissue slide at 400× magnification on a LEICA light microscope and counting the positive cells on two tissue slides per animal. Results were expressed as the number of cells per 105 μm2 tissue surface.
5
Histological Evaluation of Cartilage Degeneration
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After the macroscopic observation, the specimens were embedded in paraffin and cut into 5 μm sections. These sections were stained with haematoxylin and eosin and with safranin O for evaluation. The degenerative changes were graded by 2 blinded observers using the histological grading scale proposed by Masuda et al [31] (link) (Table 2 ).
Type II collagen expression was detected using a mouse monoclonal antibody (1∶200; Abcam, Cambridge, MA) and a horseradish peroxidase-conjugated anti-mouse antibody (1∶50; Dako, Denmark), followed by colour development with diaminobenzidine tetrahydrochloride (DAB, Dako). The results of type II collagen staining were quantified in mean density with the IPP version 6.0 software (Media Cybernetics, Bethesda, MD, USA).
Type II collagen expression was detected using a mouse monoclonal antibody (1∶200; Abcam, Cambridge, MA) and a horseradish peroxidase-conjugated anti-mouse antibody (1∶50; Dako, Denmark), followed by colour development with diaminobenzidine tetrahydrochloride (DAB, Dako). The results of type II collagen staining were quantified in mean density with the IPP version 6.0 software (Media Cybernetics, Bethesda, MD, USA).
6
Histological Evaluation of dECM-SF Scaffolds
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The samples were fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned into 5 µm sections. The sections were stained with hematoxylin and eosin (H&E) to evaluate their structures and stained with Safranin O-Fast Green to visualize glycosaminoglycan (GAG) deposits. The expression of type II collagen was detected by mouse anti-human type II collagen monoclonal antibody (1:200; Abeam, Cambridge, MA, USA), followed by horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (1:50, Dako, Denmark) and color development with diaminobenzidine tetrahydrochloride (DAB, Dako, Denmark). To detect the existence of DNA, samples of the dECM-SF scaffold and MC dECM-SF scaffold were examined under fluorescence microscopy after DAPI (4′,6-diamidino-2-phenylindole) staining. For cell viability, the scaffolds were incubated with live/dead double staining solution (Sigma, USA) for 30 min and then observed by confocal microscopy (Leica, Heidelberg, Germany).
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