Quantifying Phospho-JNK in Cervical Cancer Tissue

A cervical cancer TMA containing 39 cases of cervical cancer and 9 cases of normal cervical tissue (in duplicate) were purchased from GeneTex, Inc. (GTX21468). Slides were deparaffinised in xylene, rehydrated in a graded series of ethanol solutions and subjected to antigen retrieval in citric acid. Slides were blocked in normal serum and incubated in primary antibody (Phospho-SAPK/JNK (Thr183/Tyr185) (81E11; 4668, Cell Signalling Technology (CST))) overnight at 4 °C. Slides were then processed using the VECTASTAIN® Universal Quick HRP Kit (PK-7800; Vector Laboratories) as per the manufacturer’s instructions. Immunostaining was visualised using 3,3’-diaminobenzidine (Vector® DAB (SK-4100; Vector Laboratories)). Phospho-JNK immunostaining quantification was automated using ImageJ with the IHC Profiler plug-in [88 (link)]. Histology scores (H-score) were calculated based on the percentage of positively stained tumour cells and the staining intensity grade [89 (link)]. The staining intensities were classified into the following four categories: 0, no staining; 1, low positive staining; 2, positive staining; 3, strong positive staining. H score was calculated by the following formula: (3 × percentage of strong positive tissue) + (2 × percentage of positive tissue) + (percentage of low positive tissue), giving a range of 0–300.