On-slide straining 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 stabilizing the binding of a plurality of oligonucleotide-linked antibodies to a sample, comprising:
(a) binding the sample with a plurality of antibodies that are linked to oligonucleotides; and
(b) crosslinking the antibodies directly to the sample using a chemical crosslinker.
2. The method of claim 1 , wherein the chemical crosslinker is bifunctional cross-linker.
3. The method of claim 1 , wherein the chemical crosslinker is an amine-to-amine cross-linker.
4. The method of claim 1 , wherein the chemical crosslinker comprises formaldehyde.
5. The method of claim 1 , wherein the chemical crosslinker comprises a disuccinimidyl crosslinker.
6. The method of claim 1 , wherein the sample is planar.
7. The method of claim 1 , wherein the sample is a tissue section.
8. The method of claim 1 , wherein the sample is a formalin-fixed paraffin embedded (FFPE) tissue section.
9. The method of claim 1 , wherein the sample is a tissue biopsy.
10. The method of claim 1 , wherein the antibodies bind to diagnostic markers in the sample.
11. The method of claim 1 , wherein the plurality of antibodies comprises a first antibody that is linked to a first oligonucleotide and a second antibody that is linked to a second oligonucleotide.
12. The method of claim 1 , wherein the plurality of antibodies comprises at least 10 antibodies, each linked to a different oligonucleotide.
13. The method of claim 1 , wherein the plurality of antibodies comprises at least 50 antibodies, each linked to a different oligonucleotide.
14. The method of claim 1 , wherein the oligonucleotides are double-stranded.
15. The method of claim 1 , wherein the oligonucleotides are single-stranded.
16. The method of claim 1 , further comprising
(c) labeling some not all of the antibodies with a fluorescently label after they are cross-linked to the sample in step (b).
17. The method of claim 1 , wherein the antibodies are monoclonal antibodies.
18. The method of claim 1 , wherein the antibodies are not fluorescently labeled prior to step (b).
19. The method of claim 1 , wherein the oligonucleotides linked to the antibodies are at least 10 nucleotides in length.
20. The composition of claim 1 , wherein the oligonucleotides linked to the antibodies are in the range of 15-200 nucleotides in length.