Prolonged incubation time in immunohistochemistry: effects on fluorescence staining of immunoglobulins and epithelial components in ethanol- and formaldehyde-fixed paraffin-embedded tissues.

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Abstract
By prolonging the incubation time from 30 min to 20 hr at room temperature, fluorochrome conjugates may be applied at about ten times higher dilution and yet produce specific immunofluorescence staining of enhanced intensity. This modification of the direct method is important for reagent economy and, in addition, affords improved staining features for all antigens tested in formaldehyde-fixed tissues. It is particularly valuable when paired staining is used to characterize lymphoproliferative B-cell processes in pathological routine material; a clear-cut distinction between polyclonal and monoclonal expression of cytoplasmic immunoglobulin (Ig) is obtained, and cells giving rise to a false staining pattern are easily pinpointed. It is likewise advantageous to use prolonged incubation with conjugate for the localization of Ig-producing and Ig-bearing B cells in saline-extracted ethanol-fixed tissues, and the same holds true for Ig and C3 in immune-complex deposits. Also IgE on the surface of mast cells in tissues from atopic subjects is visualized distinctly with this modification. However, the localization or epithelial components is not consistently improved in ethanol-fixed tissues when the incubation time is prolonged; secretory products such as lactoferrin, lysozyme, amylase, and secretory component (SC) are not always immobilized sufficiently by ethanol fixation to avoid diffusion artifacts and a substantial loss from the cytoplasm. Differences in intracellular storage probably contribute to the variable antigen stability.