BACKGROUND: Aldehyde fixation is a common process used to preserve the complex structure of biological samples ex vivo. This method of fixation relies on the formation of covalent bonds between aldehydes and amines present in the biomolecules of the sample. Aldehyde fixation is routinely performed in histological studies, however fixed tissue samples are rarely used for non-histological purposes as the fixation process is thought to make brain tissue unsuitable for traditional proteomic analyses such as Western blot. Advances in antigen-retrieval procedures have allowed detectable levels of protein to be solubilized from formaldehyde fixed tissue, opening the door for aldehyde-fixed samples to be used in both histological and proteomic approaches. NEW METHOD: Here, we developed a series of antigen-retrieval steps for use on fixed-brain lysates to make them suitable for analysis by Western blot. RESULTS: Prolonged exposure of the tissue homogenate to high temperature (90 °C for 2 h) in the presence of a concentrated formaldehyde scavenger and ionic detergent was sufficient to reveal a variety of synaptic and non-synaptic proteins on membrane blots. CONCLUSION: This protocol has significant utility for future studies using fixed tissue samples in a variety of neuropathological conditions.
BACKGROUND:Aldehyde fixation is a common process used to preserve the complex structure of biological samples ex vivo. This method of fixation relies on the formation of covalent bonds between aldehydes and amines present in the biomolecules of the sample. Aldehyde fixation is routinely performed in histological studies, however fixed tissue samples are rarely used for non-histological purposes as the fixation process is thought to make brain tissue unsuitable for traditional proteomic analyses such as Western blot. Advances in antigen-retrieval procedures have allowed detectable levels of protein to be solubilized from formaldehyde fixed tissue, opening the door for aldehyde-fixed samples to be used in both histological and proteomic approaches. NEW METHOD: Here, we developed a series of antigen-retrieval steps for use on fixed-brain lysates to make them suitable for analysis by Western blot. RESULTS: Prolonged exposure of the tissue homogenate to high temperature (90 °C for 2 h) in the presence of a concentrated formaldehyde scavenger and ionic detergent was sufficient to reveal a variety of synaptic and non-synaptic proteins on membrane blots. CONCLUSION: This protocol has significant utility for future studies using fixed tissue samples in a variety of neuropathological conditions.
Authors: Aaron Y Lai; Paolo Bazzigaluppi; Christopher D Morrone; Mary E Hill; Bojana Stefanovic; JoAnne McLaurin Journal: Front Neurosci Date: 2022-06-23 Impact factor: 5.152