Protein expression was assayed by immunohistochemistry using the paraffin-embedded sections from the patients. The sections were first deparaffinized by xylene (Shanghai Sangon, Shanghai, China). Then, the sections were incubated with rabbit polyclonal anti-fibronectin antibody (1:500, ab2413; Abcam, Cambridge, MA, USA) at 4°C for 24 h. After washing with TBST buffer (Shanghai Sangon) for three times, the sections were incubated with goat anti-rabbit IgG H&L (HRP, 1:500, ab6721; Abcam) at room temperature for 1 h. Then, protein immunostaining was performed using DAB Plus substrate (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. The scoring of the stained tumor cells were divided into five grades: 0 (positive cells <5%), 1 (positive cells between 5 and 25%), 2 (positive cells between 26 and 50%), 3 (positive cells between 51 and 75%), and 4 (positive cells between 75 and 100%). The cells were observed using a light microscope (Axioskop; Zeiss, Germany). We designated grade 1 and 2 as low expression of fibronectin and grade 3 and 4 as high expression of fibronectin for the prognosis analysis.
Dab plus substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The DAB Plus Substrate is a chromogenic substrate used in immunohistochemistry and other related applications. It provides a brown staining reaction upon enzyme-mediated conversion, enabling the visualization of target proteins or other biomolecules in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Primary antibody enhancer, by Thermo Fisher Scientific (3 mentions)
Dab plus chromogen, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase polymer, by Thermo Fisher Scientific (2 mentions)
Ab2413, by Abcam (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Axioskop, by Zeiss (1 mentions)
Xylene, by Sangon (1 mentions)
Tbst buffer, by Sangon (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
S7571, by Merck Group (1 mentions)
Ultra 5 block, by Thermo Fisher Scientific (1 mentions)
Hydrogen peroxide block, by Thermo Fisher Scientific (1 mentions)
Digital imaging system, by Leica (1 mentions)
Peroxidase conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Bm purple substrate, by Roche (1 mentions)
Leukocyte acid phosphatase kit, by Merck Group (1 mentions)
Citrate buffer, by Agilent Technologies (1 mentions)
6 protocols using dab plus substrate
1
Immunohistochemical Fibronectin Expression Assay
2
Immunohistochemical Detection of ERβ in Breast Cancer
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Breast carcinoma paraffin sections were dewaxed in water and 3% H2O2 then incubated for 10 min at 20°C. The section was washed with distilled water and soaked in PBS for 5 min twice. The tissue was incubated with 5% PBS diluted goat serum (Life Technologies, Carlsbad, CA, USA) and sealed, and then incubated for 10 min at 20°C. The goat serum was discarded and the primary rabbit polyclonal ERβ antibody (dilution, 1:100; cat. no. ab60709; Abcam, Cambridge, MA, USA) was added. The tissue was incubated for 1–2 h at 37°C or stored at 4°C overnight. We added the proper amount of the working goat monoclonal biotin-labeled anti-human IgG secondary antibody (dilution, 1:1,000; cat. no. NEF803001EA; PerkinElmer, Inc., Waltham, MA, USA) to the sample and incubated it for 10 min at 37°C. HRP was added (S7571; Sigma-Aldrich, St. Louis, MO, USA), incubated for 10 min at 37°C and 1–2 drops of DAB Plus Chromogen (TA-125-HD) was added to 1 ml DAB Plus Substrate (TL-125-HDX) (both from Thermo Fisher Scientific, Waltham, MA, USA). We blended and added drops to the section, incubated the section for 10 min at 37°C, and washed and sealed the section.
3
Immunohistochemical Staining of Bone Markers
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After deparaffination and rehydrate tissue section, the specimens were washed twice in buffer (Biogear). To reduce non-spesific background staining due to endogenous peroxidase, slides were incubated in Hydrogen Peroxide Block (Thermo Scientific, USA) for 10 minutes, then washed four times in buffer (Biogear). Ultra V Block (Thermo Scientific, USA) was applied and then incubated for five minutes at room temperature to block non-spesific background staining. Primary antibody
(Runx2 monoclonal antibody SC101145 Santa Cruz Biotechnology, USA; Osterix [
EPR21034]
ab 209484, Abcam, USA) was applied and incubated at room temperature from around 25 to 30 minutes, then washed four times in buffer (Biogear). Primary Antibody Enhancer (Thermo Scientific, USA) was applied and incubated for 10 minutes at room temperature, then washed four times in buffer (Biogear). HRP Polymer (Thermo Scientific, USA) was applied and incubated for 15 minutes at room temperature then washed four times in buffer (Biogear). One drop (40µL) DAB Plus Chromogen (Thermo Scientific, USA) was added to 2 mL of DAB Plus Substrate (Thermo Scientific, USA) and mixed by swirling and applied to tissue. The substance was incubated for 5 minutes then washed 4 times in Aquabidest (PT Ikaphamindo Putramas, Jakarta, Indonesia). Counterstaining and coverslip placement was done using a permanent mounting media.
(Runx2 monoclonal antibody SC101145 Santa Cruz Biotechnology, USA; Osterix [
EPR21034]
ab 209484, Abcam, USA) was applied and incubated at room temperature from around 25 to 30 minutes, then washed four times in buffer (Biogear). Primary Antibody Enhancer (Thermo Scientific, USA) was applied and incubated for 10 minutes at room temperature, then washed four times in buffer (Biogear). HRP Polymer (Thermo Scientific, USA) was applied and incubated for 15 minutes at room temperature then washed four times in buffer (Biogear). One drop (40µL) DAB Plus Chromogen (Thermo Scientific, USA) was added to 2 mL of DAB Plus Substrate (Thermo Scientific, USA) and mixed by swirling and applied to tissue. The substance was incubated for 5 minutes then washed 4 times in Aquabidest (PT Ikaphamindo Putramas, Jakarta, Indonesia). Counterstaining and coverslip placement was done using a permanent mounting media.
4
Histological Analysis of Bone Development
5
Immunohistochemical Staining of Tumor Tissues
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For IHC, tumor tissues were extracted, fixed for 24 h in 10% formaldehyde, and embedded in paraffin. IHC staining was performed as follows. Tissue sections were deparaffinized twice with xylene for 10 min and were rehydrated using a graded alcohol series. After removing endogenous peroxidases using 0.1% H2O2, sections were washed three times with PBS. Antigen retrieval was achieved by incubating the sections in 10 mM citrate buffer (pH 6.0) (DAKO, Glostrup, Denmark) using a microwave oven. Then the sections were permeabilized with 0.5% PBX (0.5% Triton X-100 in PBS) for 30 min and washed three times with PBS. After blocking for 1 h with 5% BSA, the primary antibody was added, and the sections were incubated overnight at 4 °C. Primary Antibody Enhancer (Thermo Fisher Scientific, Waltham, MA, USA) and horseradish peroxidase Polymer (Thermo Scientific) were used for signal amplification. To develop the colored product, a mixture of DAB (3,3′-diaminobenzidine) Plus Chromogen and DAB Plus Substrate (Thermo Fisher Scientific) was added for 5 min. After washing with PBS, 20% hematoxylin counterstain was added for 2–5 min to stain the nuclei. Finally, the tissue sections were dehydrated in a graded alcohol series. After clearing twice in xylene, the tissue sections were coverslipped with mounting media (xylene:mount = 1:1) for microscopy.
6
Immunohistochemical Analysis of Tumor Tissues
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For IHC, tumor tissues of two mice per group after 7 days of third virus injection were extracted after CO2 euthanasia. Then, they were fixed for 24 h in 10% formaldehyde, and embedded in paraffin. Tissue sections were deparaffinized twice with xylene for 10 min and were rehydrated using a graded alcohol series. After antigen retrieval, the sections were permeabilized with 0.5% PBX (0.5% Triton X-100 in PBS) for 30 min and washed three times with PBS. After blocking for 1 h with 5% BSA, the sections with primary antibodies were incubated overnight at 4 °C. Primary Antibody Enhancer (Thermo Fisher Scientific) and horseradish peroxidase Polymer (Thermo Scientific) were used for signal amplification. To develop the colored product, a mixture of DAB (3,3′-diaminobenzidine) Plus Chromogen and DAB Plus Substrate (Thermo Fisher Scientific) was added for 5 min. After washing with PBS, 20% hematoxylin counterstain was added for 2–5 min to stain the nuclei. After dehydration and clearing twice in xylene, the tissue sections were coverslipped with mounting media (xylene:mount = 1:1) for microscopy.
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