A novel immunofluorescence detection method for renal cell-type specific in situ cytokine production by confocal microscopy

The detection of cytokines production in tissues is subjected to significant limitations: (1) Cytokine protein production frequently does not correlate with mRNA levels. (2) Cytokines are secreted rapidly and dissipate from the cellular source, thus making detection difficult. (3) The synthetic rate of many cytokines are low. (4) Tissue fixation ablates antigenic sites and diminishes detection signals. The identification of the cellular sources of cytokines poses an additional challenge because of the lack of suitable and readily available cellular markers. In our renal cytokine production studies in lupus nephritis, we have established methods to resolve problems associated with the identification of cellular sources of pertinent cytokines in the glomerulus and interstitium. Four-color confocal microscopy was used to colocalize cell-type specific markers with cytokines. The cytokine signal was amplified by the incubation of tissue slices in medium containing pan-specific stimulants plus secretion blockers. Tissue fixation was optimized to provide sharp crisp signals. Commercially available Ab suitable for fluorochrome labeling were used to establish cell-specific markers in the tubules and glomeruli. This combination of optimizations allowed us to define the cellular sources of important glomerular cytokines including TNF-α, IL-6, and IL-1β which appear to form a cytokine circuit in glomerulonephritis pathogenesis. ● Tissue stimulation and secretion blocking for cytokine detection ● Fixation optimization and Ab source identification for direct staining ● Colocalization of cytokines and renal cell-type specific markers.