Freshly excised tissue was fixed in 10% formalin (Sigma Aldrich, Cat#F8775) then rinsed in PBS with 0.02% sodium azide (Fisher Chemical, Cat#S227I25). The tissue was embedded in paraffin, sectioned and stained for EGFR or CLDN2 essentially as described previously.20 (link),22 (link) Rehydrated tissue was microwave boiled in Tris-EGTA buffer (TEG, 10 m m Tris, 0.5 m m EGTA, pH 9.0) for antigen retrieval. Free aldehyde groups were blocked in 0.6% H2O2 and 50 m m NH4Cl in PBS. Sections were blocked in 5% skim milk (Millipore Sigma Aldrich, Cat#70165), probed with primary antibody in 0.1% Triton-X100 in PBS overnight at 4°C and then incubated with secondary antibodies. Sections were visualized with DAB + Substrate Chromogen System (K3467, DakoCytomation) and counterstained with hematoxylin.
Protocol detail
Immunohistochemical Analysis of EGFR and CLDN2
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Aldehyde
Antibodies
Antibody
Antigen
Chromogen substrate
Egfr
Egta
Embedded paraffin
Formalin
H2o2
Hematoxylin
Microwave
Milk
Sodium azide
Tissue
Tris
Triton x100
Corresponding Organization :
Other organizations : University of Alberta, Women and Children’s Health Research Institute, Saarland University, Odense University Hospital, University of Southern Denmark
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Variable analysis
independent variables
- Tissue fixation in 10% formalin (Sigma Aldrich, Cat#F8775)
- Tissue rinsing in PBS with 0.02% sodium azide (Fisher Chemical, Cat#S227I25)
- Tissue embedding in paraffin
- Tissue sectioning
- Antigen retrieval by microwave boiling in Tris-EGTA buffer (TEG, 10 mM Tris, 0.5 mM EGTA, pH 9.0)
- Blocking of free aldehyde groups in 0.6% H2O2 and 50 mM NH4Cl in PBS
- Tissue blocking in 5% skim milk (Millipore Sigma Aldrich, Cat#70165)
- Probing with primary antibody in 0.1% Triton-X100 in PBS overnight at 4°C
- Incubation with secondary antibodies
- EGFR expression
- CLDN2 expression
- Tissue type (freshly excised tissue)
- Staining protocol (as described previously in references 20 and 22)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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