Fixation/permeabilization: New alternative procedure for immunofluorescence and mRNA in situ hybridization of vertebrate and invertebrate embryos
- Authors
- Fernandez, J., and Fuentes, R.
- ID
- ZDB-PUB-130222-2
- Date
- 2013
- Source
- Developmental Dynamics : an official publication of the American Association of Anatomists 242(5): 493-507 (Journal)
- Registered Authors
- Keywords
- fixation/permeabilization, immunofluorescence, mRNA in situ hybridization, invertebrate embryos
- MeSH Terms
-
- Animals
- Chick Embryo
- Drosophila/embryology
- Embryo, Mammalian/cytology*
- Embryo, Mammalian/metabolism
- Fluorescent Antibody Technique/methods
- In Situ Hybridization/methods*
- Leeches/embryology
- Permeability
- RNA, Messenger/analysis
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Specimen Handling/methods
- Staining and Labeling/methods
- Tissue Fixation/methods*
- Zebrafish/embryology
- PubMed
- 23389988 Full text @ Dev. Dyn.
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 non-conventional 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.