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 composition comprising:
(a) a biological sample; and
(b) a plurality of antibodies;
wherein:
i. the plurality of antibodies are linked to single-stranded oligonucleotides, and
ii. the plurality of antibodies are chemically cross-linked directly to the biological sample.
2. The composition of claim 1 , wherein the plurality of antibodies are crosslinked to the biological sample via a bifunctional cross-linker.
3. The composition of claim 1 , wherein the plurality of antibodies are crosslinked to the biological sample via an amine-to-amine crosslinker.
4. The composition of claim 1 , wherein the plurality of antibodies are crosslinked to the biological sample by formaldehyde.
5. The composition of claim 1 , wherein the plurality of antibodies are crosslinked to the biological sample by a disuccinimdyl crosslinker.
6. The composition of claim 1 , wherein the biological sample is planar.
7. The composition of claim 1 , wherein the biological sample is a tissue section.
8. The composition of claim 1 , wherein the biological sample is a formalin-fixed paraffin embedded (FFPE) tissue section.
9. The composition of claim 1 , wherein the biological sample is a tissue biopsy.
10. The composition of claim 1 , wherein the plurality of antibodies are bound to diagnostic markers in the biological sample.
11. The composition of claim 1 , wherein the plurality of antibodies comprises a first antibody linked to a first single-stranded oligonucleotide and a second antibody linked to a second single-stranded oligonucleotide.
12. The composition of claim 1 , wherein the plurality of antibodies comprises at least 10 antibodies, each linked to a different oligonucleotide.
13. The composition of claim 1 , wherein the plurality of antibodies comprises at least 50 antibodies, each linked to a different oligonucleotide.
14. The composition of claim 1 , wherein a single-stranded oligonucleotide of the single stranded oligonucleotides is linked to a second oligonucleotide strand to form a double-stranded oligonucleotide.
15. The composition of claim 14 , wherein the double-stranded oligonucleotide comprises an overhang.
16. The composition of claim 1 , wherein one or more antibodies of the plurality of antibodies are fluorescently labeled.
17. The composition of claim 16 , wherein the fluorescent label is attached to an oligonucleotide that is linked to an antibody.
18. The composition of claim 1 , wherein the plurality of antibodies are monoclonal antibodies.
19. The composition of claim 1 , wherein the single stranded oligonucleotides linked to the plurality of antibodies are at least 10 nucleotides in length.
20. The composition of claim 1 , wherein the single stranded oligonucleotides linked to the plurality of antibodies are in the range of 15-200 nucleotides in length.