Protein retention expansion microscopy
AssigneeMassachusetts Institute of Technology
InventorsPaul Warren Tillberg, Fei Chen, Edward Stuart Boyden, Chih-Chieh Yu
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Abstract
The invention provides a method termed protein retention ExM (proExM), in which proteins, rather than labels, are anchored to the swellable gel, using a cross-linking molecule. This proExM strategy can be used to perform nanoscale imaging of immunostained cells and tissues as well as samples expressing various FPs as fluorescent signals from genetically encoded fluorescent proteins and/or conventional fluorescently labeled secondary antibodies and streptavidin that are directly anchored to the gel are preserved even when subjected to the nonspecific proteolytic digestion.
Claims27
A method for the retention and imaging of proteins of a sample of interest comprising the steps of:
- (a) conjugating proteins within the sample with a bifunctional crosslinker, wherein the bifunctional crosslinker comprises a protein-reactive chemical group and a gel-reactive chemical group;
- (b) embedding the sample in a swellable material wherein proteins within the sample are anchored to the swellable material;
- (c) subjecting the sample to digestion;
- (d) swelling the swellable material to form an expanded sample;
- (e) staining the sample either before or after steps (a) (b), (c), or (d);
- and
- (f) imaging the expanded sample.
A method for the retention and imaging of proteins of a sample of interest comprising the steps of:
- (a) conjugating proteins within the sample with a bifunctional crosslinker, wherein the bifunctional crosslinker comprises a protein-reactive chemical group and a gel-reactive chemical group;
- (b) embedding the sample in a swellable material wherein proteins within the sample are anchored to the swellable material;
- (c) swelling the swellable material to form an expanded sample;
- (d) staining the sample either before or after steps (a) (b), or (c);
- and
- (e) imaging the expanded sample.
Dependencies are inferred from the claim text.