Twenty-eight ESCC samples was immunohistochemically stained by the Envision method. The sections (5 µm thick) were deparaffinized, rehydrated with graded concentrations of ethanol, and incubated with 3% H2O2 for 15 min. The sections were then incubated with the primary antibody (rabbit anti-human CHFR monoclonal antibodies, BS-4272R, Bioss, Beijing, China; mouse anti-human p16 monoclonal antibodies, ZM-0205, Zsbio, Beijing, China), followed by incubation with secondary antibodies (anti-mouse/rabbit, PV-6000, Zsbio, Beijing, China) for 15 min at room temperature and DAB reagent (ZLI-9018, Zsbio, Beijing, China).The sections were then counterstained with hematoxylin, dehydrated. CHFR, and CDKN2A (p16) staining were scored for nuclear and cytoplasmic staining based on intensity and percentage of cells stained (25 (link),26 (link)).
Protocol detail
Immunohistochemical Analysis of CHFR and p16 in ESCC
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Anti antibodies
Antibodies
Antibody
Cells
Cytoplasmic
Ethanol
H2o2
Hematoxylin
Human
Monoclonal antibodies
Mouse
Rabbit
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Variable analysis
independent variables
- Staining method (Envision method)
- Nuclear and cytoplasmic staining intensity and percentage of cells stained for CHFR and CDKN2A (p16)
- Section thickness (5 µm)
- Incubation time with primary antibody (unknown)
- Incubation time with secondary antibody (15 min)
- Incubation time with H2O2 (15 min)
- Room temperature
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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