4.10. Immunohistochemistry (IHC) Staining Assay

Serial sections (2 μm) were cut from the paraffin blocks and mounted on pre-coated slides for immunohistochemical analysis of the CAM tumors. All FFPE CAM tissue sections were deparaffinized with xylene and rehydrated with graded ethanol. The antigen retrieval was performed by 1 min steam cooking in TRS-Buffer pH 6 (p21 Waf1/Cip1) or pH 9 (pChk2^T68), respectively. Slides were incubated at 4 °C overnight with a primary monoclonal antibody p21 Waf1/Cip1 (12D1, mouse), 1:1.000 dilution (Cell Signaling), or pChk2^T68 (2197, rabbit), 1:50 dilution (Cell Signaling). Antibody binding was visualized using the Polymer-Kit (AP, Zytomed, Berlin, Germany) or biotinylated Anti-Rabbit/ABC-Kit (Vector, Olean, NY, USA).

Adjacent slides were stained with hematoxylin and eosin for histomorphological analyses. Immunohistochemical scoring was performed in a semi-quantitative way by two pathologists (KEW, AVR). The intensity was quantified in a range from 1 to 3. The area was quantified by percentage of positive cells in 5% steps. An immunoscore was generated by multiplying intensity (0–3) with the respective percentage of positive cells (0–100%).

Bright field images (Magnification: 200×, 400×) of stained sections were taken with the Olympus XC50 camera (Olympus Corporation, Tokyo, Japan) in combination with the Olympus BX51 microscope (Olympus Corporation).