Double Labeling Fluorescent Immunocytochemistry

Fluorescent immunocytochemistry is a powerful technique based on detecting antigens. It leads to discoveries in cell composition and structure as well as its functioning by expanding knowledge on colocalization between its components. The potency of this method is based on findings in the areas of specific antibodies production, fluorescent labels, and microscopy. Since it merges different fields, it requires basic knowledge on all the steps that are needed in the procedure planning and implementation to be used properly and produce reliable results. Here we describe a protocol of LN-229 human glioblastoma cells double labeling of LC3 and IRS-1 proteins, highlighting the importance of some steps of the procedure and possible variables.