Optimisation of immunocytochemistry methodology for the detection of endogenous eIF2B localised foci

Abstract

The multisubunit eukaryotic initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF) for eIF2, is an essential regulator of translation initiation. Activation of the cellular integrated stress response (ISR) by factors such as endoplasmic reticulum stress leads to phosphorylation of eIF2α and inhibition of eIF2B GEF activity. Cytoplasmic bodies containing eIF2B subunits, termed eIF2B bodies, have been shown to alter in subunit composition and fluorescence recovery after photobleaching activity in response to the ISR. Analysis of the subunit composition of endogenous eIF2B bodies is dependent on accurate detection of each protein in a cellular context via immunocytochemistry (ICC). We describe bioinformatic techniques to optimize the ICC detection of eIF2B foci in U373 cells. The screening of commercially available primary antibodies against predicted epitopes enhanced measurements of the number, size and fluorescence intensity of eIF2B bodies. A consistent and reproducible ICC analysis of endogenous eIF2B bodies will aid characterisation of eIF2B bodies during the ISR or under disease conditions.