Immunohistochemical staining was performed on normal, diseased autopsied d-HCM and hypertensive human hearts, the explanted heart of the patient undergoing cardiac transplantation for advanced d-HCM and spheres of iPSC-induced cardiomyocyotes using a labeled streptavidin-biotin method (DAKO Corp., Santa Clara, CA, USA). Left ventricular free wall samples from the human heart were formalin-fixed and paraffin embedded. Deparaffinized 3-μm-thick sections were heated for 5 min at 100°C in a pressure cooker to reactivate the antigen, then treated with 0.3% H2O2 in methanol for 30 min to abolish endogenous peroxidase activity. Sections were blocked with 10% goat serum in PBS, incubated with the primary antibody (anti-GJB4 or anti-GJA1 rabbit antibody (Abcam)) overnight at 4°C, washed, covered with second-step biotinylated antibody for 30 min, and incubated with peroxidase-labeled streptavidin for 30 min. After washing, sections were then incubated with 0.05% diaminobenzidine/0.15% H2O2 and counterstained with 10% hematoxylin. Image acquisition was done with a BZ-X700 microscope (Keyence, Osaka, Japan).
Labeled streptavidin biotin method
Manufactured by Agilent Technologies
Sourced in United States, Denmark, Japan
The Labeled streptavidin–biotin method is a technique used to detect and quantify specific biomolecules in a sample. Streptavidin, a protein derived from the bacterium Streptomyces, has a high affinity for biotin, a small molecule. In this method, a target biomolecule is labeled with biotin, and the presence of the target is detected by the binding of a streptavidin-labeled marker, such as a fluorescent dye or an enzyme. The strength of the streptavidin-biotin interaction allows for sensitive and specific detection of the target molecule.
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Lab products found in correlation
Bz x700 microscope, by Keyence (1 mentions)
Anti rabbit alexa594 antibody, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti p53 antibody, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Anti zo 1, by Abcam (1 mentions)
Anti pcna antibody, by Santa Cruz Biotechnology (1 mentions)
Anti muc2, by GeneTex (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
6 protocols using labeled streptavidin biotin method
1
Immunohistochemical Analysis of Cardiac Gap Junctions
2
Immunohistochemical Analysis of Phosphorylated α-Synuclein
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The brain tissues were fixed in 20% neutral buffered formalin, and 8-μm paraffin sections were prepared on glass slides with a microtome. After deparaffinization and rehydration, the tissue sections were subjected to staining with hematoxylin and eosin, and immunohistochemical staining using anti-Ser129-phosphorylated αSyn antibody (Cat. No. 015-25191; Wako, RRID: AB_2537218, 1:3000). To enhance the immunogenicity, the sections were prepared by heating for 40 min at 98 °C prior to incubation with primary antibody. Primary antibody binding was detected by the labeled streptavidin-biotin method (DAKO). Peroxidase-conjugated streptavidin was visualized with 3′3-diaminobenzidine (Cat. No. 7411-49-6; Wako) as the chromogen. Immunostained sections were lightly counterstained with Mayer’s hematoxylin.
3
Immunohistochemical Analysis of sCJD
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Paraformaldehyde-fixed, formic-acid treated, 4-μm thick paraffin-embedded sections of sCJD cases were obtained with a sliding microtome. The sections were incubated with 2% hydrogen peroxide and 10% methanol for 30 min at room temperature, followed by 5% normal serum for 2 h. Then the sections were incubated overnight with one of the primary antibodies. After washing, the sections were processed with the labeled streptavidin–biotin method (Dako). Some sections were stained without the primary antibody or with secondary antibody alone to rule out non-specific immunoreactivity. Tissue sections were slightly counterstained with hematoxylin.
4
Immunohistochemical Staining of Brain Tissue
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The brain tissues were fixed in 20% neutral buffered formalin, embedded in paraffin, cut into Sects. 8 μm thick with a microtome, and placed on glass slides. After deparaffinization and rehydration, the tissue sections were heated for 40 min at 98 °C for antigen retrieval. All sections were immersed in hydrogen peroxide (0.3%) solution for 10 min to quench endogenous peroxidase activity and incubated with specific primary antibodies (1:100) diluted in PBS containing 1% BSA for 2 h. Primary antibody binding was detected by the labeled streptavidin–biotin method (DAKO). Peroxidase-conjugated streptavidin was visualized with 3′3-diaminobenzidine (7411-49-6; Wako) as the chromogen. Immunostained sections were lightly counterstained with Mayer’s hematoxylin.
5
Immunohistochemical Analysis of Tissue Markers
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We used a Tris‐EDTA buffer (pH 9.0) antigen retrieval method (for 10 minutes at 110°C) for all immunohistochemical staining. The primary antibodies used were as follows: anti‐p53 mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐active caspase‐3 rabbit polyclonal antibody (Cell Signaling Technology, Danvers, MA, USA), anti‐podoplanin hamster monoclonal antibody (AngioBio, Del Mar, CA, USA) and anti‐CD31 rabbit polyclonal antibody (Lab Vision, Fremont, CA, USA). We incubated the slides with corresponding biotinylated secondary antibodies using an established labeled streptavidin‐biotin method (Dako, Glostrup, Denmark), with exposure to diaminobenzidine and hematoxylin counterstain for visualizing the immunocomplexes formed. In addition, anti‐phospho‐Akt‐Ser473/Thr308 rabbit polyclonal antibody (Cell Signaling Technology) and anti‐rabbit Alexa‐594 antibody (Thermo Fisher Scientific, Waltham, MA, USA) were used for immunofluorescence staining. Nuclear staining was conducted with DAPI (Vector Labs, Burlingame, CA, USA); phospho‐Akt‐positive areas and viable regions with DAPI‐positive nuclei in the same field were digitally captured at 40× magnification. These digitized images were measured using the ImageJ program (NIHR public domain) and the results are expressed as a percentage of phospho‐Akt‐positive areas to nuclear‐positive areas.
6
Evaluating Cell Proliferation and Apoptosis in Colonic Tumors
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Using the labeled streptavidin–biotin method (DAKO, Kyoto, Japan), immunohistochemical staining for PCNA, a marker for the G1-to-S phase, and cleaved caspase-3 was performed to examine cell proliferation and apoptosis, respectively, in the colonic tumours26 (link),49 (link). Immunohistochemical staining for MUC2 and ZO1 in the colonic mucosa was performed to evaluate mucosal barrier function. Anti-PCNA antibody (1:100, Santa Cruz Biotechnology, Dallas, TX, USA), anti-cleaved caspase-3 antibody (1:500, Ser276; Cell Signaling Technology, Danvers, MA, USA), anti-MUC2 (GeneTex, Irvine, CA, USA), and anti-ZO1 (abcam, Cambridge, UK) were used. PCNA-positive cells and cleaved caspase-3-stained cells were counted and expressed as a percentage of the total number of cells in the colonic tumours. The positive rate was determined by counting at least 200 cells in each mouse. Immunohistochemically stained areas for MUC2 and ZO1 were evaluated and quantified with NIH Image software (Bethesda, MD, USA) according to the section of tutorials and examples in the website of the software (https://imagej.nih.gov/ij/docs/examples/stained-sections/index.html ). The quantitative analysis was performed with four images from each mouse in a blinded manner.
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