AbstractThe histological effects of freezing and thawing unfixed tissue before experimental diffusion studies across the mucosal barrier were investigated in the face of claims that such treatment was of little significance. Following fixation, tissue sections were stained by periodic acid-Schiff (PAS), alcian blue (pH 1.0&2.5)-PAS, Masson and Mallory trichromes, acid-picro-Mallory phosphotungstic acid-hematoxylin, Martius-scarlet-blue, luxol-fast-blue, and Sudan black B. Trichrome type staining revealed a previously unrecorded artifact, particularly evident in quenched tissues. Three distinct epithelial cell types could be identified on the basis of differential dye uptake. This finding was not evident in PAS, alcian blue, or Sudan black B stained sections. We postulate that distortion of the cellular matrix occurs as a result of the freeze, thaw, diffusion, and fix sequence and that these cells illustrate the well known phenomenon of tissue density/dye molecular size differential staining by acid dyes. (The J Histotechnol16:343, 1993)