On-slide staining by primer extension
A method for analyzing planar sample is provided. In some cases the method comprises: (a) labelling the planar sample with a capture agent that is linked to a nucleic acid, wherein the capture agent specifically binds to complementary sites in the planar sample; (b) reading a fluorescent signal caused by extension of a primer that is hybridized to the nucleic acid, using fluorescence microscopy. Several implementations of the method, and multiplexed versions of the same, are also provided.
1. A method for analyzing a planar sample, the method comprising:
(a) labeling the planar sample with a capture agent to produce a labeled sample,
wherein
the capture agent is linked to a nucleic acid comprising an overhang sequence;
and
(ii) the capture agent comprises an antibody;
(b) contacting the labeled sample with a labeled oligonucleotide, wherein the labeled oligonucleotide comprises an fluorescent dye optical label and hybridizes to at least a portion of the overhang sequence,
(c) contacting the labeled sample with a ligase, thereby ligating the labeled oligonucleotide to the nucleic acid; and
(d) reading a signal generated by the labeled oligonucleotide, wherein the reading comprises fluorescent microscopy.
2. The method of claim 1 , wherein the capture agent is linked to the nucleic acid via a covalent link.
3. The method of claim 2 , wherein the covalent link is produced via click chemistry.
4. The method of claim 1 , after (a), further comprising cross-linking the capture agent to the planar sample.
5. The method of claim 1 , wherein the optical label comprises a fluorescent dye.
6. The method of claim 1 , wherein the fluorescent dye is a cyanine dye.
7. The method of claim 1 , wherein the cyanine dye is selected from the group consisting of Cy3, Cy5, and Cy7.
8. The method of claim 1 , wherein the capture agent comprises an antibody.
9. The method of claim 1 , further comprising staining the planar sample with a cytological stain after (d).
10. The method of claim 9 , wherein the cytological stain comprises DAPI, hematoxylin, eosin, or a combination thereof.
11. The method of claim 1 , further comprising staining the planar sample with a cytological stain before (a).
12. The method of claim 11 , wherein the cytological stain comprises DAPI, hematoxylin, eosin, or a combination thereof.
13. The method of claim 1 , wherein the planar sample is a formalin-fixed, paraffin-embedded (FFPE) section.
14. The method of claim 1 , wherein the ligase is a T4 ligase.
15. The method of claim 1 , after (d), further comprising inactivating the signal generated by the labeled oligonucleotide.