Because PLA can detect endogenous proteins, and requires few cells, we decided to broaden PLA technology to include the identification of fusion proteins and protein interactions in hematological malignant cells. For that purpose, we chose to optimize the detection of ETV6-RUNX1 fusion protein and the interaction between RUNX1 and CBFB, its well-known cofactor, in non-adherent pre-B lymphoblasts. A flowchart of the successive steps of the optimized PLA protocol is presented in Fig. 1. Briefly, the cells are cytospun, fixed, permeabilized, and incubated with two primary antibodies raised in different species. Then, we performed hybridization with PLA probes, ligation of the probes, rolling circle amplification, slide mounting, image acquisition by microscopy and image analysis (Fig. 1).Fig. 1Optimized protocol outlines of Proximity Ligation Assay for non-adherent pre-B lymphoblasts. Schematic outline summarizing the procedure of PLA for detection, visualization and quantification of individual endogenous proteins, protein modifications and protein interactions. Asterisk depicts steps that have been adapted from the original manufacturer’s procedure