Deparaffinized tissue slides were blocked with 3% H2O2 solution and antigen were retrieved with 10 mM citrate buffer (pH 6.0). After blocking, appropriately diluted primary antibodies were added onto the slides and incubated in a humidified chamber at 4 °C overnight, and then appropriately diluted biotinylated secondary antibody was incubated at room temperature for 1 hr. DAB substrate solution (Dako, K5361) (freshly made just before use) was used to reveal the color of antibody staining. Nuclei were localized by heamtoxylin staining for 1–2 min before mounting and capture.
Dab substrate solution
Manufactured by Agilent Technologies
Sourced in United States
The DAB substrate solution is a laboratory reagent used in immunohistochemistry and similar analytical techniques. It provides a chromogenic substrate for the detection of target antigens or proteins in biological samples. The solution produces a brown color reaction when catalyzed by the enzyme-labeled detection system, allowing for the visualization and localization of the target of interest.
Automatically generated - may contain errors
Lab products found in correlation
Inpp4b, by Cell Signaling Technology (1 mentions)
Goat serum, by Abcam (1 mentions)
Ab170904, by Abcam (1 mentions)
Las software, by Leica (1 mentions)
Alpha smooth muscle actin α sma, by Agilent Technologies (1 mentions)
Vascular endothelial growth factor (vegf), by Santa Cruz Biotechnology (1 mentions)
Dako envision system, by Agilent Technologies (1 mentions)
5 protocols using dab substrate solution
1
Immunohistochemical Staining Protocol
2
Immunohistochemical Analysis of Epithelial Markers
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Paraffin sections (4 μm) were treated with 3% H2O2 and blocked with serum-blocking reagent for 10 min each at room temperature; sections were then incubated with primary antibody (keratin 15, 1:200 dilutions, Santa Cruz, sc-56520; keratin 19, 1:200 dilutions, Santa Cruz, sc-53258; CEA, 1:100 dilutions, Cell SIGNALING, #2383) overnight at 4 °C. Sections were washed with PBS and incubated with secondary antibody (1:100 dilutions, Santa Cruz) for 30 min at room temperature, washed with PBS, incubated with 100 μl GTVision ChemMate Envision HRP (DAKO) at room temperature for 30 min and developed with DAB substrate solution (DAKO KIT freshly made just before use) to reveal the color of antibody staining. All sections were then examined and photographed with Zeiss KS400 image analysis system (Carl Zeiss, Oberkochen, Germany).
3
INPP4B Immunohistochemical Staining
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EMP histological sections were deparaffinized and blocked with 3% H2O2 solution, and antigen was retrieved with 10 mM citrate buffer (pH 6.0). After blocking, appropriately diluted primary INPP4B (#14543, Cell Signaling Technology) antibodies were added onto the slides and incubated in a humidified chamber at 4°C overnight, and then, appropriately diluted biotinylated secondary antibody was incubated at room temperature for 1 h. 3,3′-Diaminobenzidine (DAB) substrate solution (Dako, K5361) (freshly made just before use) was used to reveal the color of antibody staining. Nuclei were localized by hematoxylin staining for 1–2 min before mounting and capture.
4
Immunohistochemical Detection of P-glycoprotein in Placental Tissue
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Five µm thick paraffin-embedded sections of placental tissue were dewaxed and rehydrated before antigen retrieval. Endogenous tissue peroxidase activity was blocked in 3% hydrogen peroxide in ethanol, and permeability was obtained by 0.5% Triton X100 in PBS. Blocking was performed by using 10% normal goat serum (Cat. No. 10000C, Thermo Fisher, Life Technologies, Denmark) in PBS for 30 min. Sections were incubated overnight at 4 °C with primary anti-P-glycoprotein antibody (ab170904, Abcam, Denmark) in a 1:200 dilution in PBS containing 10% goat serum. A negative control was incubated in PBS containing 10% goat serum only. After washing, sections were incubated in 1:400 diluted HRP-conjugated goat anti-rabbit secondary antibody (Cat. No. 7074, Cell Signaling, Denmark). Sections were developed with DAB substrate solution (Dako, USA) and counterstained with hematoxylin. Images were captured by using Leica microscope (DMI400B) and Leica LAS software (Leica Microsystems, Limited Version 4.11.0, Switzerland).
5
Immunohistochemical Analysis of Vascular Markers
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Paraffin sections were de-waxed in xylene, rinsed in grade alcohol, and rehydrated in water. Then they were placed in citric buffer (pH 6.0) and treated in a microwave oven with high power for 3 minutes and subsequent low power for 10 minutes. Afterwards, the sections underwent blocking with 3% peroxidase for 20 minutes and 10% goat serum for 30 minutes. Subsequently, primary antibodies with proper dilution were applied on the sections, which were then incubated at 4°C overnight. Following that, secondary antibodies from Dako EnVision™ System (DakoCytomation, Glostrup, Denmark) were applied, and the sections were incubated for 30 minutes at room temperature. Signals were developed with DAB substrate solution (DakoCytomation). The sections were finally counter-stained by hematoxylin solution. Primary antibodies used in this study included VEGF (Santa Cruz Biotechnology), alpha smooth muscle actin (α-SMA, DakoCytomation), CD34 (Santa Cruz Biotechnology), and CD68 (BD Biosciences, San Jose, CA, USA).
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