A Cost-Effective Workflow for Mono- and Multiplex Immunofluorescence in Adherent Two-Dimensional Cell Cultures on Glass Coverslips.

Immunofluorescence (IF) microscopy is a widely used technique for visualizing protein localization and co-distribution at single-cell resolution while preserving cellular morphology and subcellular organization. The goal of this protocol is to present a practical and cost-effective workflow for mono- and multiplex immunofluorescence staining of adherent 2D cell cultures using standard laboratory materials. The method relies on culturing cells on inexpensive glass coverslips placed in conventional 24-well plates and implements multiplexing through host-species separation of primary antibodies, spectrally distinct fluorophores, and robust control conditions, including no-primary, no-antibody, single-stain, and crosstalk controls to ensure interpretability of multiplex IF. Low-volume staining steps are used to reduce reagent and antibody consumption without compromising signal quality. This workflow avoids the need for specialized imaging plates or chambers and is compatible with routine widefield or confocal fluorescence microscopy. Overall, this protocol provides an accessible and practical immunofluorescence workflow for representative assessment of protein localization and co-distribution in adherent cell cultures. Adaptation to other cell types or antibody panels requires target-specific optimization, including antibody validation, fixation and permeabilization conditions, fluorophore selection, and compatibility with microscope and filter sets.