The tumour tissues were fixed in 4% paraformaldehyde for 24 h, followed by dehydrating in a graded alcohol series and embedding in paraffin. The tissues were cut into 5μm sections. The sections were deparaffinised, rehydrated with a graded alcohol series and then incubated in 96°C with 0.01 mol/L sodium citrate buffer for the antigen retrieval. Following the incubation in 5% H2O2 for a period of 2 h, the sections were incubated using anti-ki67 antibody overnight at 4°C. Immunostaining was carried out with the use of streptavidin–peroxidase and diaminobenzidine (DAB) following the manufacturer’s instructions (Beyotime, Shanghai, China). Eventually, the sections were not only observed under a fluorescence microscope (Leica, Wetzlar, Germany) but also imaged.
Streptavidin peroxidase
Manufactured by Beyotime
Sourced in China
Streptavidin-peroxidase is a conjugate of streptavidin and horseradish peroxidase. Streptavidin has a strong affinity for biotin, and the peroxidase enzyme can catalyze a colorimetric reaction. This product is commonly used in various immunoassay techniques and as a detection reagent in biochemical and molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Uorescence microscope, by Leica (3 mentions)
Diaminobenzidine dab, by Beyotime (2 mentions)
Diaminobenzidinef dab, by Beyotime (2 mentions)
Fluorescence microscope, by Leica (1 mentions)
Anti ptbp2 antibody, by Abcam (1 mentions)
Biotin labeled secondary antibody, by Beyotime (1 mentions)
Dab substrate kit, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Beyotime (1 mentions)
9 protocols using streptavidin peroxidase
1
Immunohistochemistry of Ki-67 in Tumour Tissues
2
Immunohistochemical Analysis of Ki67 Expression
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The tumor tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated in a graded alcohol series and embedded in paraffin followed by cutting into 5 μm sections. The sections were deparaffinized, rehydrated with a graded alcohol series, and then incubated in 96℃ with 0.01 mol/L sodium citrate buffer for the antigen retrieval. After incubation in 5% H2O2 for 2 h, the sections were incubated with Ki67 primary antibodies overnight at 4℃. Immunostaining was performed using streptavidin‐peroxidase and diaminobenzidine (DAB) according to the manufacturer's instructions (Beyotime). Finally, the sections were observed under a fluorescence microscope (Leica) and pictured.
3
Evaluating Testicular Ptbp2 Protein Localization
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To assess the degree of damage to the sperm cells, hematoxylin and eosin (HE) staining was carried out to evaluate testicular histological changes in the mouse model. In addition, localization of the Ptbp2 protein in the testicular tissues of mice was determined by immunohistochemistry (IHC). The testicular samples were fixed with 4% paraformaldehyde, then embedded in paraffin wax and sectioned at 4 μm. The testicular tissue sections were deparaffinized in different concentrations of dimethylbenzene and ethanol solutions, then heated in sodium citrate buffer (pH 6.0) in a microwave for 20 min. Next, the sections were dipped in deionized water containing 3% H2O2 to quench endogenous peroxidase activity. After specific treatment with 10% normal goat serum to block non-specific binding, the deparaffinized sections were incubated overnight at 4°C with the anti-PTBP2 antibody (Abcam) at 1:300 dilution, then incubated with a goat anti-rabbit biotinylated secondary antibody for 40 min at room temperature. The immunoreactivity with Ptbp2 was observed using streptavidin-peroxidase and 3,3N-diaminobenzidine (Beyotime, Jiangsu, China).
4
Immunohistochemical Staining of Formalin-Fixed Paraffin-Embedded Tissues
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The tissue specimens were first formalin (10%) fixed and paraffin-embedded. After that, the slides were deparaffinized at 55°C for 20 minutes followed by three washes with xylene, and then rehydrated with graded ethanol with concentrations at 100%, 95%, and 80%. The endogenous peroxidase activity was blocked by 3% hydrogen peroxide, and antigen retrieval was achieved by heating in citrate buffer (pH=6.0) in a microwave oven for 10 minutes. After being incubated in primary antibody dilution (1:100) at 4°C overnight, the slides were then incubated for 30 minutes each in a biotin-labeled secondary antibody (Beyotime Institute of Biotechnology, Shanghai, People’s Republic of China), followed by incubation in streptavidin-peroxidase (Beyotime Institute of Biotechnology). The visualization of slides was achieved with a 3,3′-diaminobenzidine substrate. Finally, the sections were counterstained with hematoxylin, dehydrated, and mounted. In the IHC test, the negative control was the sample with phosphate buffered saline (PBS) incubation instead of primary antibody, with all the other procedures remaining the same, while positive control was placental tissue sections, which had high FGF1 expression.14 (link),15 (link)
5
Immunohistochemical Analysis of ZNF143 in Liver Tissue
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Liver tissues were fixed with 4% paraformaldehyde (Sigma-Aldrich) at room temperature (RT) for more than 24 h, and embedded in paraffin. Tissue sections were deparaffinized with xylene and rehydrated with graded series of ethanol (absolute, 95%, 90%, 80%, 75%, respectively, and distilled water), followed by wash twice with PBS-T for 5 min. Antigen retrieval was performed for 10 min in 10 mM sodium citrate buffer (pH 6.0) at 95–100 °C followed by wash with PBS-T for 5 min at RT. Immediately, tissue sections were incubated in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. Tissue sections were then washed with PBS-T for 5 min and blocked (Immunostaining Blocking Solution: 2% normal goat serum, 2% bovine serum albumin (BSA) and 0.1% Triton-X in PBS) for 30 min at RT. Tissue sections were then incubated in humidified chamber for over 16 h at 4 °C with ZNF143 primary antibody (Proteintech, 16,618–1-AP) (1:100 in TBST). Tissue sections were washed three times with PBS-T for 5 min and incubated at RT for 1 h with secondary antibody (goat anti rabbit). After wash twice with PBS-T for 5 min at RT, sections were incubated with streptavidin peroxidase (Beyotime) for 10 min at RT and the color was developed using a DAB substrate kit (Beyotime).
6
Immunohistochemical Analysis of Breast Cancer Tissues
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Breast cancer tissues were fixed in 10% neutral buffered formalin before being embedded in paraffin. The tissues were then cut into 6-micron thickness with a microtome. The slides were deparaffinized and rehydrated before antigen retrieval as described previously [20 (link)]. All slides were then subject to blocking in 10% normal serum for 10 minutes, followed by incubation with antibodies against RNF6, ERα, PR or HER2, overnight at 4°C. A biotin-conjugated secondary antibody diluted with Tris-based buffer (TBS) containing 10% serum and 1% BSA was applied to incubate for 10 minutes. After rinsing with cold TBS, the slides were then incubated with Streptavidin-peroxidase for 10 minutes before being stained with 3,3′-Diaminobenzidine (Beyotime, Nantong, China). The slides were finally stained with Hematoxylin and eosin before being mounted for microscopy analysis.
7
Immunohistochemical Analysis of Tumor Tissues
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The tumor tissues were xed in 4% paraformaldehyde for 24 h, followed by dehydrating in a graded alcohol series and embedding in para n, followed by cutting into 5 µm sections. The sections were depara nized, rehydrated with a graded alcohol series and then incubated in 96 ℃ with 0.01 mol/l sodium citrate buffer for the antigen retrieval. Following the incubation in 5% H2O2 for a period of 2 h, the sections were incubated using primary antibodies overnight at 4 ℃. Immunostaining was carried out with the use of streptavidin-peroxidase following the manufacturer's instructions (Beyotime, Shanghai, China).
Finally, the sections were not only observed under a uorescence microscope (Leica, Wetzlar, Germany) but also imaged. Each trial was conducted at least 3 times.
Finally, the sections were not only observed under a uorescence microscope (Leica, Wetzlar, Germany) but also imaged. Each trial was conducted at least 3 times.
8
Immunohistochemical Analysis of Tumor Tissues
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The tumour tissues were xed in 4% paraformaldehyde for 24 hours, followed by dehydrating in a graded alcohol series and embedding in para n, followed by cutting into 5μm sections. The sections were depara nised, rehydrated with a graded alcohol series and then incubated in 96℃ with 0.01 mol/l sodium citrate buffer for the antigen retrieval. Following the incubation in 5% H 2 O 2 for a period of 2 hours, the sections were incubated using primary antibodies including ki67 and SMAD1 (Abcam, England) overnight at 4℃. Immunostaining was carried out with the use of streptavidin-peroxidase and diaminobenzidinef (DAB) following the manufacturer's instructions (Beyotime, Shanghai, China). Eventually, the sections were not only observed under a uorescence microscope (Leica, Wetzlar, Germany) but also imaged.
9
Immunohistochemical Analysis of Tumor Tissues
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Tumor tissues were xed in 4% paraformaldehyde for 24 hours, and then dehydrated in alcohol, embedded in para n, and cutted into 5μm sections. The sections were depara nized, rehydrated, and then incubated at 96℃ with 0.01 mol/l sodium citrate buffer for the antigen retrieval. Following the incubation in 5% H 2 O 2 for 2 hours, the sections were incubated with primary antibodies overnight at 4℃.
Immunostaining was carried out with streptavidin-peroxidase and diaminobenzidinef (DAB) following the manufacturer's instructions (Beyotime, Shanghai, China). Finally, the sections were observed and imaged under uorescence microscope (Leica, Wetzlar, Germany).
Immunostaining was carried out with streptavidin-peroxidase and diaminobenzidinef (DAB) following the manufacturer's instructions (Beyotime, Shanghai, China). Finally, the sections were observed and imaged under uorescence microscope (Leica, Wetzlar, Germany).
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