Quantifying TP53 Isoforms in Tumors

Probes included a custom probe to the unique region of ∆133TP53 and TP53β made by Advanced Cell Diagnostics named ∆133TP53 and described elsewhere [2 (link),3 (link)], as well as the probes ubiquitin C (UBC, positive control) and DapB (negative control, Advanced Cell Diagnostics, Newark, CA, USA). Formalin-fixed paraffin-embedded tumors or cell clots were cut into 5 µm sections. The RNAscope method used the manual assay method with Protease Plus reagent for protein digestion and the 2.5HD reagent kit brown according to the manufacturer’s instructions. Following the addition of DAB to the ∆133TP53 and DapB assays, DAB enhancer was added (Leica Biosystems, Wetzlar, Germany). Positive cells and the number of dots per positive cell were identified using the Aperio Scancope CS digital pathology system and quantified using the Aperio RNA ISH Algorithm (Aperio, Vista, CA, USA). The slides were evaluated by two blinded examiners. The percentage of positive cells out of the total cell number was measured. Ten fields (×400 magnification) were chosen at random and the percentage of positive cells out of the total cells counted. Tumors with no positive cells in the 10 random fields were examined over the entire tissue section for the presence of positive cells.