The construction process and detailed information on the HEC tissue microarray (TMA) and immunohistochemistry (IHC) process have been described previously 24 (link),25 (link).Briefly, the slides were deparaffinized and rehydrated in xylene and alcohol gradient, then treated with citric acid epitope retrieval reagent at 100°C for 20 min and cooled to room temperature to prohibit the endogenous peroxidase activity. To block nonspecific binding sites, the sections were incubated with 5% bovine serum albumin (BSA) (YESEN, Shanghai, China) at 37˚C for 30 min. Subsequently, the sections were incubated with primary rabbit anti‑human monoclonal H3K4me3 antibody (#9751, 1:200 dilution, Cell Signaling Technology) overnight at 4°C. The sections were washed with PBS three times and then incubated for 1 hours with horseradish peroxidase (HRP)-labeled secondary antibody (Gene Tech; Shanghai, China). Finally, the sections were stained using diaminobenzidine (DAB) (Gene Tech; Shanghai, China) and imaged by using a microscope (Leica Microsystems Imaging Solutions, Cambridge, UK).
Horseradish peroxidase hrp labeled secondary antibody
Manufactured by Gene Tech
Sourced in China
Horseradish peroxidase (HRP)-labeled secondary antibody is a laboratory reagent used to detect and visualize target proteins in various experimental techniques, such as Western blotting and immunohistochemistry. The HRP enzyme is conjugated to the secondary antibody, which binds to the primary antibody directed against the target protein. This facilitates the detection of the target protein through a chromogenic or chemiluminescent reaction.
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4 protocols using horseradish peroxidase hrp labeled secondary antibody
1
Histone H3K4me3 Immunohistochemistry Protocol
2
Immunohistochemical Staining Protocol
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IHC was performed as previously described [20 (link)]. Briefly, the slides were deparaffinised in xylene and rehydrated in graded ethanol. After incubation in 0.3% H2O2, antigen retrieval was performed in citrate buffer. Subsequently, sections were incubated with the primary antibody (Additional file 1 : Table S1), horseradish peroxidase (HRP)-labeled secondary antibody (Gene Tech, Shanghai, China), stained with diaminobenzidine (DAB, Gene Tech, Shanghai, China), counterstained with hematoxylin, then, dehydrated in ethanol, cleared in xylene, and cover-slipped with resin. The integrated optical density (IOD) value was assessed by Image Pro Plus software (V 6.0).
3
Immunohistochemical Analysis of CD11b+ Cells
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The paraffin-embedded slides were dewaxed, rehydrated, and incubated in 3% H2O2 for 20 min and calf serum for 15 min, respectively. Next, the slides were incubated with anti-CD11b antibody (Servicebio, Wuhan, China) at 4°C overnight, before being incubated with horseradish peroxidase (HRP)-labeled secondary antibody (Gene Tech, Shanghai, China). After washing with PBS, the slides were stained with DAB and hematoxylin, respectively. Finally, eight fields per slide were randomly photographed in order to assess and quantify CD11b-positive cells using ImageJ software.
4
Tissue Microarray Construction and Immunohistochemistry
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Detailed information concerning the tissue microarray (TMA) construction was provided in our previous report 22 (link). Briefly, the TMA included 85 cases of squamous carcinoma, 110 adenocarcinomas and 13 other types of NSCLC. Following dewaxing at 60°C for 2 h and rehydration, the paraffin sections were blocked using 3% H2O2 for 15 min to inactivate endogenous peroxidase activity. Subsequently, the sections were incubated in citric acid epitope retrieval reagent at 100°C for 20 min and allowed to cool to room temperature. To block nonspecific binding sites, the sections were treated with 5% bovine serum albumin (BSA) (YESEN, Shanghai, China) for 1 h. Then, the sections were incubated with primary antibodies overnight at 4°C. The primary antibodies were rabbit anti-human RNF38 (25132-1-AP; 1:100 dilution; Proteintech) and rabbit anti-human E-cadherin (EP700Y, 1:500 dilution, Epitomics). The slides were incubated with a horseradish peroxidase (HRP)-labeled secondary antibody (Gene Tech, Shanghai, China) for 1 h at room temperature. After washing off the residual secondary antibody that had not bound the primary antibody, the sections were stained with diaminobenzidine (DAB, Gene Tech, Shanghai, China) and observed under a microscope.
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