Specimens from CRC patients and mice were formalin fixed, sectioned, and embedded into paraffin for immunohistochemistry. Immunofluorescence was performed with frozen samples and freshly fixed cells. The following antibodies were used: anti-CD8 (1:500), anti-CXCL10 (1:300), anti-CD4 (1:300), anti-IL-17A (1:300), and anti-CXCR3 (1:300; all Abcam, Cambridge, UK). Immunohistochemistry and immunofluorescence were performed as described elsewhere [33 (link), 34 (link)]. For immunohistochemistry, three fields of view per sample were imaged. The intensity of immunostaining was considered when analyzing the data. The percentage scoring of immunoreactive cells was as follows: 0 (< 10%), 1 (10–40%), 2 (40–70%), and 3 (> 70%). Staining intensity was visually scored and stratified as follows: 0 (negative), 1 (yellowish), 2 (light brown), and 3 (dark brown). Immunoreactivity scores (IRS) were obtained by multiplying the two items to a total score and ranged from 0 to 9. Protein expression levels were further analyzed by classifying IRS values as low (based on an IRS value ≤ 5) and as high (based on an IRS value > 5).
Free full text: Click here
