For H&E staining and IHC analysis, FFPE blocks were cut into 5-μm sections. For IHC, sections were blocked with normal horse or goat serum (Vector) for 1 h and then incubated overnight at 4 °C with the following antibodies in CAS-Block (Invitrogen): AXIN2 (1:200, Abcam), CD44 (1:200, eBioscience), CKIα (1:1,000, Abcam) Cyclin D1 (1:125, Thermo Fisher Scientific), Ki67 (1:200, Neomarkers), p21 (1:50, Santa Cruz Biotechnology), p53 (1:200, Novocastra), PROX1 (1:200, R&D Systems) and SOX9 (1:1,000, Abcam). Secondary antibodies were horseradish peroxidase (HRP) polymer-conjugated goat anti-rat, horse anti-rabbit, horse anti-goat (Vector) or anti-mouse (mouse-on-mouse; Nichirei). Diaminobenzidine (DAB) chromogen (Thermo Fisher Scientific) was used for detection and haematoxylin was used as a counterstain. Images were taken with a BX51 Olympus microscope. Measurement of the number and size of tumours and the degree of dysplasia in histology sections was performed blindly.
Chromogen diaminobenzidine dab
Manufactured by Thermo Fisher Scientific
Sourced in United States
Chromogen diaminobenzidine (DAB) is a colorimetric substrate used in immunohistochemistry and in-situ hybridization techniques to detect the presence of specific target molecules in biological samples. It produces a brown-colored precipitate at the site of the target, which can be visualized under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Normal horse or goat serum, by Vector Laboratories (2 mentions)
Cas block, by Thermo Fisher Scientific (2 mentions)
Cyclin d1, by Thermo Fisher Scientific (2 mentions)
Axin2, by Abcam (2 mentions)
Bx51 microscope, by Olympus (2 mentions)
Prox1, by R&D Systems (2 mentions)
Horseradish peroxidase hrp polymer conjugated goat anti rat, by Vector Laboratories (2 mentions)
Horse anti goat, by Vector Laboratories (2 mentions)
Anti mouse mouse on mouse, by Vector Laboratories (2 mentions)
Ultra 5 block, by Thermo Fisher Scientific (2 mentions)
Gfap antibody, by Thermo Fisher Scientific (1 mentions)
Entellan, by Merck Group (1 mentions)
E cadherin antibody, by Thermo Fisher Scientific (1 mentions)
7 protocols using chromogen diaminobenzidine dab
1
Histological and Immunohistochemical Analysis of Tumors
2
Histological and Immunohistochemical Analysis of Tumors
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For H&E staining and IHC analysis, FFPE blocks were cut into 5-μm sections. For IHC, sections were blocked with normal horse or goat serum (Vector) for 1 h and then incubated overnight at 4 °C with the following antibodies in CAS-Block (Invitrogen): AXIN2 (1:200, Abcam), CD44 (1:200, eBioscience), CKIα (1:1,000, Abcam) Cyclin D1 (1:125, Thermo Fisher Scientific), Ki67 (1:200, Neomarkers), p21 (1:50, Santa Cruz Biotechnology), p53 (1:200, Novocastra), PROX1 (1:200, R&D Systems) and SOX9 (1:1,000, Abcam). Secondary antibodies were horseradish peroxidase (HRP) polymer-conjugated goat anti-rat, horse anti-rabbit, horse anti-goat (Vector) or anti-mouse (mouse-on-mouse; Nichirei). Diaminobenzidine (DAB) chromogen (Thermo Fisher Scientific) was used for detection and haematoxylin was used as a counterstain. Images were taken with a BX51 Olympus microscope. Measurement of the number and size of tumours and the degree of dysplasia in histology sections was performed blindly.
3
Immunohistochemical Analysis of Testis Tissue
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Formaldehyde-fixed testis tissue was embedded in paraffin wax for further immunohistochemical examination. Sections were deparaffinized in xylene. The antigen retrieval process was performed twice in citrate buffer solution (pH 6.0), first for 7 minutes, and then for 5 minutes, and was boiled in a microwave oven at 700 W. They were then allowed to cool to room temperature for 30 minutes and washed twice in distilled water for 5 minutes. Endogenous peroxidase activity was blocked in 0.1% hydrogen peroxide for 20 minutes. Ultra V block (Cat. No. 85-9043, Invitrogen, Carlsbad, California, USA) was applied for 10 minutes prior to the application of primary antibodies for Bcl-2 (cat: PA5-20068, Invitrogen, Carlsbad, California, USA) and VEGF (cat: PA5-16754, Invitrogen, Carlsbad, California, USA). Secondary antibody (Cat. No. 85-9043, Invitrogen, Carlsbad, California, USA) was applied for 20 minutes. Slides were then exposed to streptavidin-peroxidase for 20 minutes. Chromogen diaminobenzidine (DAB, Cat. No. 34002, Invitrogen, Carlsbad, California, USA) was used. Control slides were prepared as mentioned above, but by omitting the primary antibodies. After counter staining with hematoxylin and washing in tap water for 8 minutes and in distilled water for 10 minutes, sections were examined using a light microscope (Zeiss, Germany).
4
Immunohistochemical Analysis of Tissue Sections
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Formaldehyde-fixed tissueswere embedded in paraffin wax for further IHC examination. Sections were deparaffinized in absolute alcohol. Antigen retrieval process was performed twice in citrate buffer solution (pH:6.0), first for 7 minutes, and second for 5 minutes, 90℃×3 minutes in the microwave waited. boiled in a microwave oven at 700 W. They were allowed to cool at room temperature for 30 minutes and washed twice in distilled water for 5 minutes. Endogenous peroxidase activity was blocked in 0.1% hydrogen peroxide for 20 minutes. Ultra V block (Cat.No: 85-9043; Invitrogen, Carlsbad, CA, USA) was applied for 10 minutes prior to the application of primary antibodies VEGF antibody (dilution rate, 1/100), cluster of differentiation 68 (CD68) antibody (dilution rate, 1/100) and Phosphorylation p38 MAPK Antibody (dilution rate, 1/100) overnight. Secondary antibody (Cat.No: 85-9043; Invitrogen) was applied for 20 minutes. Slides were then exposed to streptavidin-peroxidase for 20 minutes. Chromogen diaminobenzidine (DAB; Invitrogen) was used. Control slides were prepared as mentioned above, but omitting the primary antibodies. After counterstaining with hematoxylin, and washing in tap water for 8 minutes and holding in distilled water for 10 minutes, the slides were mounted with entellan.
5
Immunohistochemical Analysis of Tissue Sections
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Formaldehyde-fixed tissue was embedded in paraffin wax for further immunohistochemical examination. Sections were deparaffinised in xylene. Antigen retrieval process was performed twice in citrate buffer solution (pH: 6.0), first for 7 min, and second for 5 min, boiled in a microwave oven at 700 W. They were allowed to cool to room temperature for 30 min and washed twice in distilled water for 5 min. Endogenous peroxidase activity was blocked in 0.1% hydrogen peroxide for 20 min. Ultra V block (Cat. No:85-9043, Invitrogen, Carlsbad, CA, USA) was applied for 10 min prior to the overnight application of primary antibodies VEGF antibody (Cat. No: RB-222-P0) (1:100 dilution), GFAP antibody (1:100 dilution) (Cat. No: PA3-067, Invitrogen, Carlsbad, CA, USA). Secondary antibody (Cat. No: 85-9043, Invitrogen, Carlsbad, CA, USA) was applied for 20 min. Slides were then exposed to streptavidin-peroxidase for 20 min. Chromogen diaminobenzidine (DAB) (Invitrogen, Cat. No: 34002 Carlsbad, CA, USA) was used. Control slides were prepared as mentioned above, but omitting the primary antibodies. After counterstaining with haematoxylin, and washing in tap water for 8 min and in distilled water for 10 min, the slides were mounted with Entellan.
6
Immunohistochemical Analysis of Tissue Markers
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Formaldehyde-fi xed tissue was embedded in paraffi n for immunohistochemical examination. The sections were deparaffi nised in absolute alcohol. Antigen retrieval was performed twice in citrate buffer solution (pH 6.0), fi rst for 7 minutes and then for 5 minutes, and boiled in a microwave oven at 700 W. The specimens were allowed to cool to room temperature for 30 minutes and were then washed twice in distilled water, for 5 minutes each time. Endogenous peroxidase activity was blocked with 0.1 % hydrogen peroxide for 20 minutes. Ultra V block (Cat. no. 85-9043; Invitrogen, Carlsbad, CA, USA) was applied for 10 minutes prior to overnight application of the primary antibodies: ET-1 antibody (1:100; Invitrogen), NF-κB antibody (1:100; Invitrogen), and ADAM-15 antibody (1:100; Invitrogen). Secondary antibody (Cat. no. 85-9043; Invitrogen) was applied for 20 minutes. Then the slides were exposed to streptavidin-peroxidase for 20 minutes. Chromogen diaminobenzidine (DAB, Cat. No. 34002, Invitrogen) was used. Control slides were prepared as described above, omitting the primary antibodies. After counterstaining with haematoxylin, and washing the slide in tap water for 8 minutes and in distilled water for 10 minutes, the slides were mounted with Entellan (Cat. no. 107961; Sigma-Aldrich, St. Louis, MO, USA).
7
Immunohistochemical Analysis of E-Cadherin and VEGF
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Formaldehyde-fixed tissue was embedded in paraffin wax for further immunohistochemical examination. Sections were deparaffinised in absolute alcohol. The antigen retrieval process was performed twice in citrate buffer solution (pH 6.0), first for 7 min, and second for 5 min, boiled in a microwave oven at 700 W. They were allowed to cool to room temperature for 30 min and washed twice in distilled water for 5 min. Endogenous peroxidase activity was blocked in 0.1% hydrogen peroxide for 20 min. Ultra V block (Cat. No. 85-9043, Invitrogen, Carlsbad, California, USA) was applied for 10 min prior to the application of primary antibodies E-Cadherin antibody 1:100 (Cat. No. MA5-12023, Invitrogen), Vascular-Endothelial Factor (VEGF) antibody (1:100) (Cat. No. PA3-067, Invitrogen). Secondary antibody (Cat. No. 85-9043, Invitrogen) was applied for 20 min. Slides were then exposed to streptavidin-peroxidase for 20 min. Chromogen diaminobenzidine (DAB, Cat. No. 34002, Invitrogen) was used. Control slides were prepared as mentioned above, but omitting the primary antibodies. After counterstaining with Haematoxylin and washing in tap water for 8 min and in distilled water for 10 min. Then, sections were examined in light microscope.
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