Fixation/Permeabilization: New alternative procedure for immunofluorescence and mrna in situ hybridization of vertebrate and invertebrate embryos

摘要

A new procedure is described to visualize the spatial pattern of expression of proteins and mRNAs in cryosections or whole-mounted leech, Drosophila, zebrafish, and chick embryos. Our principal contribution is in the use of a nonconventional fixation/permeabilization procedure based on the use of formaldehyde or paraformaldehyde combined with a short C-chain carboxylic acid. Detergents, methanol, and proteinases were omitted. Hybridization procedures were modified from those of routinely used protocols developed for the same embryos. Results showed that cytoskeletal and other cytoplasmic proteins, as well as different mRNAs, were clearly visualized in the expected regions of the embryos. Our procedure has several advantages over currently used protocols: is simpler, produces better general preservation of cells, yields reliable results, and can be used for embryos of different taxa at different developmental stages. It is hypothesized that short C-chain aliphatic carboxylic acids modulate the cross-linking effect of aldehyde fixatives on cell proteins.