For immunohistochemistry (IHC), sections were deparaffinized and hydrated. Serial slides were used for PCNA (Proliferating Cell Nuclear Antigen) staining. Tissue sections were treated with 0.1% triton X-100 (Sigma-Aldrich) in PBS 1x (Life Technologies) at room temperature (RT) for 10 min. Then endogenous peroxidases were inhibited by incubation with 3%H2O2 in methanol at RT for 10 min. Tissue sections were then placed in an antigen retrieval solution (0.01 M citrate buffer, pH = 6 (Zytomed, Berlin, Germany) for 15 min at 350 W) and quenched for endogenous peroxidases as described above. After saturation (GBI labs, Washington, USA), mouse anti-PCNA (M0879, DakoCytomation, Courtaboeuf, France) at 525 µg/ml was applied for 1 h at 37 °C. Sections were incubated with a kit anti-mouse HRP (GBI labs) for 30 min at RT. Staining was developed with HRP Green (Zytomed). Then, sections were counterstained with nuclear fast red (H3403, VectorLabs, Burlingame, CA, USA), dehydrated and mounted. Isotype control antibodies are used as negative controls.
Citrate buffer
Manufactured by Zytomed Systems
Sourced in Germany
Citrate buffer is a widely used aqueous solution that maintains a specific pH range, typically between 3.0 and 6.2, depending on the concentration of the buffer components. This buffer solution is commonly used in various laboratory applications, including biological assays, protein purification, and pH adjustments.
Automatically generated - may contain errors
2 protocols using citrate buffer
1
PCNA Immunohistochemistry in Tissue Sections
2
Immunohistochemical Analysis of Liver Samples
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Liver samples were fixed with 4% paraformaldehyde in phosphate-buffered saline and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin at the Seoul National University Hospital Biomedical Research Institute facility. Immunohistochemical staining with an anti-preS1 monoclonal antibody (Aprogen, Daejeon, South Korea) was also performed. Deparaffinized sections were heated in citrate buffer (Zytomed, Berlin, Germany) to accomplish antigen retrieval. Endogenous peroxidase was blocked with peroxidase blocking solution (Zytomed). An anti-preS1 antibody was applied as the primary antibody followed by the application of the avidin-biotin complex method to detect the primary antibody. Peroxidase activity was visualized by a 3,3’-diaminobenzidine substrate kit (Zytomed) with hematoxylin (Wako, Osaka, Japan) as counterstain.
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