SERIAL DETECTION OF MULTIPLE TARGETS

1 . A method of detecting multiple biomolecules, said method comprising:

(a) contacting a cell or tissue with a first probe and a second probe, thereby forming a first complex and a second complex, wherein

the first complex comprises the first probe bound to a first biomolecule, wherein said first probe comprises a first detectable label attached to the first probe via a first linker;

the second complex comprises the second probe bound to a second biomolecule, wherein said second probe comprises a second detectable label and a quenching moiety, wherein said second detectable label is attached to the second probe via a second linker; and said quenching moiety is attached to the second probe via a first cleavable linker;

(b) detecting the first complex; cleaving the first cleavable linker, thereby separating the quenching moiety from the second complex; and

(c) detecting the second complex.

2 . The method of claim 1 , wherein the first detectable label generates a first signal and the second detectable label generates a second signal, wherein said first signal and second signal are the same.

3 . The method of claim 1 , wherein the first linker and the first cleavable linker are each a chemically cleavable linker, enzymatically cleavable linker, photo-cleavable linker.

4 . The method of claim 1 , wherein the first linker and the first cleavable linker are each a polynucleotide sequence comprising a restriction site.

5 . The method of claim 1 , further comprising contacting the cell or tissue with a third probe and forming a third complex, wherein the third complex comprises the third probe bound to a third biomolecule, wherein said third probe comprises a third detectable label and a quenching moiety, wherein said third detectable label is attached to the third probe via a third linker; and said quenching moiety is attached to the third probe via a second cleavable linker;

and the method further comprises cleaving the second cleavable linker; and

detecting the third complex.

6 . The method of claim 1 , further comprising repeating steps (a)-(c).

7 . The method of claim 1 , wherein said first probe is a specific binding reagent capable of binding to a first biomolecule and the second probe is a specific binding reagent capable of binding to a second biomolecule, wherein the first biomolecule and the second biomolecule are different, and wherein the specific binding reagent is an antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), affimer, or an aptamer.

8 . The method of claim 1 , wherein said first probe is an oligonucleotide capable of binding to a first biomolecule and the second biomolecule is an oligonucleotide capable of binding to a first target, wherein the first biomolecule and the second biomolecule are different.

9 . The method of claim 1 , wherein said first biomolecule and second biomolecule are in a cell.

10 . A kit comprising:

(i) a first compound having the formula: Ab1-L 1 -Dye1;

(ii) a second compound having the formula: Ab2-L 2 -Dye2-L 1 -Q 1 ;

(iii) a third compound having the formula: Ab3-L 3 -Dye3-L 2 -Q 2 ;

wherein,

Ab1 is a first antibody; Ab2 is a second antibody; Ab3 is a third antibody;

L 1 , L 2 and L 3 are orthogonally cleavable linkers;

Dye 1, Dye 2, and Dye 3 are independently fluorescent moieties;

Q 1 and Q 2 are quenching moieties, wherein Dye 2 and Q1 are a first fluorescent-quencher pair; and Dye 3 and Q2 are a second fluorescent-quencher pair.

11 . The kit of claim 10 , wherein Dye 1, Dye 2, and Dye 3 each comprise the same fluorescent moiety.

12 . The kit of claim 10 , wherein said kit is stored at 2-8° C.

13 . A method of detecting multiple biomolecules, said method comprising:

(a) contacting a cell or tissue with a first probe and a second probe, thereby forming a first complex and a second complex, wherein

the first complex comprises the first probe bound to a first biomolecule, wherein said first probe comprises a first detectable label attached to the first probe via a first cleavable linker;

the second complex comprises the second probe bound to a second biomolecule, wherein said second probe comprises a second detectable label, wherein said second detectable label is attached to the second probe via a second cleavable linker;

(b) detecting the first complex and cleaving the first cleavable linker, thereby separating the first detectable label from the first complex;

(c) detecting the second complex and cleaving the second cleavable linker, thereby separating the second detectable label from the second complex.

14 . The method of claim 13 , further comprising repeating steps (a) and (b) for a third biomolecule.

15 . The method of claim 13 , further comprising contacting the cell or tissue with a stain, wherein the stain binds to a third biomolecule.

16 . The method of claim 13 , wherein the stain is a fluorescent stain.

17 . The method of claim 16 , wherein detecting comprises directing an excitation light to the cell or tissue and detecting an emission light from the first fluorescent dye, the second fluorescent dye, and the stain.

18 . The method of claim 13 , wherein the first biomolecule and the second biomolecule each independently a different organelle.

19 . A cell, comprising:

a first organelle bound to a first probe, wherein said first probe comprises a specific binding agent bound to the first organelle and a fluorescent dye linked to the specific binding agent via a first cleavable linker;

a second organelle bound to a second probe, wherein said second probe comprises a specific binding agent bound to the second organelle and a fluorescent dye linked to the specific binding agent via a second cleavable linker; and

a fluorescent stain bound to a nucleic acid molecule.

20 . The cell of claim 19 , further comprising a third organelle bound to a third probe, wherein said third probe comprises a specific binding agent bound to the third organelle and a linker remnant covalently attached to the specific binding agent.