Image analysis to quantify histological and immunofluorescent staining of ex vivo skin and skin cell cultures

Image processing steps and analysis techniques were developed for the quantification of photomicrographs obtained from light and fluorescence microscopy. The substrates examined were either skin cell cultures, such as normal human keratinocytes (NHK) or fibroblasts, or ex vivo skin sections. Examples of the analyses are provided for the comparison of skincare active ingredient treated samples vs. placebo to demonstrate the utility of the methods to quantify and provide numerical data for a procedure that is typically qualitative in nature and based on observations by a histologist. Quantifiable experiments that are discussed include: Fontana Masson staining for melanin expression; Nile red staining to detect cellular lipid droplets; nuclei staining with diamidino-phenylindole (DAPI); and immunofluorescent staining of protein expression with a primary antibody directed against the protein (antigen) and a secondary antibody tagged with a fluorescent dye (Alexa Fluor 488) against the primary antibody.