IHC for target molecules was performed on serial sections from tumor tissues of nude mice xenografts and CRC patients. Tissue sections were deparaffinized, subjected to antigen retrieval using target antigen retrieval solution (Dako), and incubated with primary antibodies against Ki67 (Dako M7240), cleaved Caspase-3 (CST #9661), β-catenin (BD Biosciences #610154), GATA6 (Abcam #22600), DKK1 (Abcam #61034), DKK3 (Abcam #115869), ZNRF3 (Abcam #122353), RNF43 (Abcam #129401), and APC2 (Abcam #113370). Then sections were incubated with Envision System HRP-labeled polymer anti-rabbit or anti-mouse secondary antibodies (Dako). The results of IHC were scored by two independent observers. Ki-67 and cleaved Caspase-3 staining was quantified by calculating positively stained cells in at least 5 randomly chosen HPFs of each sample. Quantification of other molecules was based on intensity and extent of staining according to the histological scoring method as previously described68 (link).
Protocol detail
Immunohistochemical Profiling of Xenograft and CRC Tissues
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target antigen retrieval solution
Envision System HRP-labeled polymer anti-rabbit or anti-mouse secondary antibodies
Anti antibodies
Antibodies
Antigen
Apc2
Caspase 3
Cells
Dkk3
Histological method
Mouse
Nude mice
Patients
Polymer
Rabbit
Tissue
Tumor
Xenografts
β catenin
Corresponding Organization : Vanderbilt University Medical Center
Other organizations : Xiamen University, Vanderbilt University, Moffitt Cancer Center, Medizinische Hochschule Hannover
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Variable analysis
independent variables
- Tissue sections
- Primary antibodies against Ki67, cleaved Caspase-3, β-catenin, GATA6, DKK1, DKK3, ZNRF3, RNF43, and APC2
- Ki-67 and cleaved Caspase-3 staining quantification
- Intensity and extent of staining for other molecules
- Tumor tissues of nude mice xenografts and CRC patients
- Deparaffinization of tissue sections
- Antigen retrieval using target antigen retrieval solution (Dako)
- Incubation with Envision System HRP-labeled polymer anti-rabbit or anti-mouse secondary antibodies (Dako)
- Scoring by two independent observers
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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