On-slide staining by primer extension
A method for analyzing planar sample is provided. In some cases the method comprises: (a) incubating the planar sample with a capture agent that is linked to an oligonucleotide, 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 oligonucleotide, using fluorescence microscopy. Several implementations of the method, and multiplexed versions of the same, are also provided.
1. A method for imaging a sample, the method comprising:
(a) incubating the sample with a plurality of capture agents under conditions in which the plurality of capture agents specifically bind to complementary sites in the sample and wherein different oligonucleotides are linked to the plurality of capture agents;
(b) treating the sample with a chemical crosslinker to crosslink capture agents in the plurality of capture agents directly to the sample;
(c) subjecting the sample to one or more cycles of:
(i) labeling one or more capture agents in the plurality of capture agents with one or more labels;
(ii) reading fluorescence signals generated by the labeling of (i); and
(iii) inactivating or removing the one or more labels of (i).
2. The method of claim 1 , wherein the sample is a planar sample.
3. The method of claim 1 , wherein the sample comprises cells grown on a planar surface, a tissue section, or a formalin-fixed paraffin embedded (FFPE) tissue section.
4. The method of claim 1 , wherein the plurality of capture agents is selected from the group consisting of: antibodies, aptamers, or oligonucleotide probes.
5. The method of claim 1 , wherein the different oligonucleotides linked to the capture agents in the plurality are in the range of 15 to 200 nucleotides in length.
6. The method of claim 1 , wherein the different oligonucleotides linked to the capture agents in the plurality are in the range of 30 to 200 nucleotides in length.
7. The method of claim 1 , wherein the inactivating in (iii) comprises photobleaching, peroxide-based bleaching, inactivation by ozone, cleavage of a fluorophore linked to an oligonucleotide through a cleavable linker, base-exchange by an exo+ polymerase, or subsequent incorporation of a quencher.
8. The method of claim 1 , wherein each of the capture agents in the plurality is linked to a different oligonucleotide.
9. The method of claim 1 , wherein each capture agent of the plurality is linked to a 5′ end of an oligonucleotide.
10. The method of claim 1 , wherein the chemical crosslinker is a bifunctional cross-linker.
11. The method of claim 1 , wherein the chemical crosslinker is an amine-to-amine cross-linker.
12. The method of claim 1 , wherein the chemical crosslinker comprises formaldehyde.
13. The method of claim 1 , wherein the chemical crosslinker comprises a disuccinimdyl crosslinker.
14. The method of claim 1 , wherein the fluorescence signals in (ii) are read using fluorescence microscopy.
15. The method of claim 1 , wherein, in each cycle of the multiple cycles, different one or more capture agents are labeled, read, and inactivated.
16. The method of claim 1 , wherein the plurality of capture agents comprises antibodies.
17. The method of claim 1 , wherein subjecting the sample to one or more cycles in (c) produces at least one image showing a pattern of binding of at least some of the plurality of capture agents to the sample.