Home › Cell Biology › Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer
Cell Biology JoVE (Open Access) Citable · DOI

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

DOI: 10.3791/1931-v
What you'll learn
  • ✓ Identify Pms2 and ERCC1 deficiency patterns in colonic field defects using immunostaining
  • ✓ Prepare and process colon tissue sections for protein expression analysis
  • ✓ Evaluate immunostained tissue sections to assess DNA repair protein distribution
  • ✓ Recognize field defect biomarkers associated with colorectal cancer progression
Protocol

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

Difficulty
advanced
Total time
~4–6 hours per tissue sample (including tissue preparation, immunostaining, and evaluation)
Model organism
Mouse (colon tissue from adenocarcinoma-bearing models)
Biosafety
BSL-1

Steps

1
Prepare colonic tissue sections for analysis

Collect and prepare colon tissue samples, including regions adjacent to adenocarcinomas. Perform sectioning and mounting on slides for downstream immunostaining.

▶ 03:04
2
Perform immunostaining on tissue sections

Apply antibodies against Pms2, ERCC1, Ku86, and CcOI proteins to detect expression patterns in colonic crypts. Include appropriate positive/negative controls and incubation steps.

▶ 04:15
3
Evaluate immunostained tissue sections microscopically

Examine stained sections at high magnification to assess protein expression levels within crypts, identify field defects with reduced/absent Pms2 or ERCC1, and quantify abnormalities in regions flanking adenocarcinomas.

▶ 08:16
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