The tumor, lung, liver, and kidney were excised from the mice, fixed in 10% neutral formalin, embedded in paraffin, and cut into 4‐μm‐thick sections. At least 10 randomly selected tissue sections from each group were stained with HE for histopathological analysis. The tumor tissue sections were incubated in 3% hydrogen peroxide for 15 min and then in normal goat serum for 20 min to block endogenous peroxidase activity and unspecific binding sites, respectively. Sections were then incubated for 2 h with primary rabbit antibodies against DNMT1, RASSF1A, APC, MST1/2, MOAP1, CNK1, cyclin D1, β‐catenin, and TUNEL (1:100 dilution) (KeyGen Biotechnology). Sections were then treated with biotinylated secondary antibodies (1:200 dilution), and antibody‐binding sites were visualized by 3,3′‐diaminobenzidine staining (Dako, Carpinteria, CA, USA). Sections were observed under an inverted phase contrast microscope (IX51; Olympus, Tokyo, Japan). Photographs were taken and analyzed using Image‐Pro Plus software (Media Cybernetics, Inc, Rockville, MD, USA).
3 3 diaminobenzidine staining
Manufactured by Agilent Technologies
Sourced in United States
3,3′‐diaminobenzidine (DAB) is a chromogenic substrate used in immunohistochemistry and other biological assays to detect the presence of specific target molecules. When enzymatically oxidized, DAB forms a brown, insoluble precipitate at the site of the target, allowing for visual identification and localization.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated secondary antibody, by Agilent Technologies (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Mouse anti mcl 1, by Santa Cruz Biotechnology (1 mentions)
Ckx41 inverted microscope, by Olympus (1 mentions)
3 protocols using 3 3 diaminobenzidine staining
1
Histopathological Analysis of Tumor Tissues
2
Immunohistochemical Analysis of Apoptosis Regulators
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Paraffin-embedded tissue sections were hydrated using a descending alcohol series, incubated for 10–20 min in target retrieval solution (DAKO, Glostrup, Denmark) and incubated with blocking solution (2% normal goat serum/PBS). After permeabilization, sections were incubated overnight at 4 °C with mouse-anti Mcl-1 (1/200; Santa Cruz, Dallas, TX, USA), mouse-anti Bcl-x (1/50, BD Bioscience, Heidelberg, Germany), or mouse-anti- Hypoxyprobe™ Mab-1 antibody for detection of pimonidazole (Hydroxyprobe, Burlington, MA, USA). Antigens were detected with a horseradish peroxidase-conjugated secondary antibody (1/250) and 3,3′-diaminobenzidine staining (DAKO) as previously described [48 , 49 (link)]. Nuclei were counterstained using hematoxylin.
3
Immunohistochemical Analysis of Tumor Markers
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The tumor excised from in vivo experiment were fixed in 10% neutral formalin, embedded in paraffin, and cut into 4-μm thick sections. The tumor tissue sections were incubated in 3% hydrogen peroxide for 15 min and then in normal goat serum for 20 min to block endogenous peroxidase activity and nonspecific binding sites. Sections were incubated for 4 h with antibodies against DNMT1, DNMT3a, DNMT3b, E-cadherin, and TIMP3 (1:100 dilution), then treated with biotinylated secondary antibodies (1:200 dilution), and antibody-binding sites were visualized by 3, 3'-diaminobenzidine staining (Dako, Carpinteria, CA, USA). Sections were observed under an Olympus CKX 41 inverted microscope.
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