LAUSR

Freeze-fracture replica immunogold labeling (FRIL) is a high-resolution immunocytochemical technique for visualizing, identifying, and mapping intramembrane proteins (IMPs) to ultrastructurally-identified membrane domains in complex tissues. Although light microscopic immunocytochemistry (LMIC) allows simultaneous use of different fluorophores bound to antibodies from multiple animal species, allowing separate detection, quantification, and subcellular mapping of multiple immunologically-distinct proteins over histological dimensions (Fig. 1A), spatial resolution in LMIC is limited by wavelength of the fluorophores to 100-300 nm. Moreover, detection of labeled proteins in conventional 5-10 μm-thick tissue slices is limited to ≥100 molecules, limited primarily by detection of florescence “signal” above autofluorescence “noise” [1]). Consequently, multiple “controls” are required for all LMIC investigations. In addition to confirmation of specificity of labeling of purified proteins in Western blots, additional “controls” required: 1) demonstration that similar fluorescent labeling did not occur in specimens in which the target protein had been knocked out (ko control), and 2) documentation that all labeling was abolished when the primary antibody was omitted while including the secondary antibodies (control for non-specific binding of secondary antibodies on other tissue components).