Methods of analyzing an H and E stained biological sample

1. A method of analyzing a hematoxylin and eosin stained biological sample, comprising:

(a) optionally detecting hematoxylin and eosin staining of a biological sample containing multiple targets and stained with hematoxylin and eosin;

(b) removing fluorescent hematoxylin and eosin signals from the biological sample; and

(c) detecting additional features or targets in the biological sample;

wherein removing the fluorescent hematoxylin and eosin signals from the biological sample comprises:

(1) washing the biological sample using a buffered aqueous solution to remove the hematoxylin and partially remove the eosin; and contacting the biological sample with a charge transfer reagent to quench residual fluorescence;

(2) washing the biological sample using a buffered aqueous solution to remove the hematoxylin and partially remove the eosin; contacting the biological sample with hydrogen peroxide and a metal salt to generate hydroxyl radicals; and optionally irradiating the biological sample; or

(3) washing the biological sample using a buffered aqueous solution to remove the hematoxylin and partially remove the eosin; contacting the biological sample with an electron transfer reagent; and irradiating the biological sample to remove residual fluorescence.

2. The method of claim 1 , wherein removing the fluorescent hematoxylin and eosin signals from the biological sample includes a wash using a buffered aqueous solution.

3. The method of claim 1 , wherein removing the fluorescent hematoxylin and eosin signals from the biological sample comprises

(i) washing the biological sample using a buffered aqueous solution to remove the hematoxylin and partially remove the eosin; and

(ii) contacting the biological sample with a charge transfer reagent to quench the residual fluorescence.

4. The method of claim 1 , wherein removing the fluorescent hematoxylin and eosin signals from the biological sample comprises

(i) washing the biological sample using a buffered aqueous solution to remove the hematoxylin and partially remove the eosin;

(ii) contacting the biological sample with hydrogen peroxide and a metal salt to generate hydroxyl radicals; and

(iii) optionally irradiating the biological sample.

5. The method of claim 1 , wherein removing the fluorescent hematoxylin and eosin signals from the biological sample comprises

(i) washing the biological sample using a buffered aqueous solution to remove the hematoxylin and partially remove the eosin;

(ii) contacting the biological sample with an electron transfer reagent; and

(iii) irradiating the biological sample to remove residual fluorescence.

6. The method of claim 5 , wherein irradiating the biological sample is accomplished by exposing the biological sample to light of 350 nm-1.3 μM in wavelength.

7. The method of claim 6 , wherein irradiating the biological sample is accomplished by exposing the biological sample to light of 400-700 nm in wavelength.

8. The method of claim 5 , wherein irradiating the biological sample is carried out in the presence of a buffer at pH of 5-9.

9. The method of claim 5 , wherein irradiating the biological sample is carried out at a temperature of 4-50° C.

10. The method of claim 5 , wherein irradiating the biological sample is performed for about 20 seconds to about 15 minutes.

11. The method of claim 5 , wherein the eosin is irreversibly modified.

12. The method of claim 5 , wherein no detectable signal is detected after biological sample irradiation.

13. The method according to claim 5 , wherein the electron transfer reagent is a borate salt.

14. The method of claim 13 , wherein the borate salt is represented by the following structural formula:

wherein:

each R 1 , R 2 , and R 3 is, independently, an alkyl, an alkenyl, an akynyl, an aryl or a heteroaryl, wherein said alkyl, alkenyl, alkynyl, aryl or heteroaryl is optionally substituted with one or more substituents selected from the group consisting of (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkylamino, amino, hydroxyl, cyano, halogen, or nitro,

R 4 is an alkyl, an alkenyl, or an akynyl, wherein said alkyl, alkenyl, or alkynyl is optionally substituted with one or more substituents selected from the group consisting of (C1-C4)alkyl, aryl, (C1-C4)alkoxy, (C1-C4)alkylamino, amino, hydroxyl, cyano, halogen, or nitro, and

M + is selected from the group consisting of organic and inorganic cations.

15. The method of claim 14 , wherein each of R 1 , R 2 , and R 3 is each independently an optionally substituted aryl and R 4 is an optionally substituted alkyl.

16. The method of claim 15 , wherein each of R 1 , R 2 , and R 3 is phenyl and R 4 is butyl, or benzyl.

17. The method of claim 14 , wherein M + is an inorganic cation selected from the group consisting of Li + , Na + , and K + .

18. The method of claim 1 , wherein detecting additional features or targets in the biological sample comprises:

(i) binding at least one probe to one or more targets of the biological sample;

(ii) detecting a signal from the at least one bound probe; and

(iii) optionally removing the signal and optionally repeating steps (i) through (iii).

19. The method of claim 18 , wherein removing the signal in step (iii) three includes exposure to a photoactivated chemical bleaching agent and irradiating the biological sample by exposing the sample to light of 350 nm-1.3 μM in wavelength.

20. The method of claim 18 , wherein the at least one probe in step (i) comprises a fluorescent signal generator, and the signal detected in step (ii) is a fluorescent signal.

21. The method of claim 18 , wherein removing the signal in step (iii) comprises contacting the biological sample with a bleaching or stripping agent or subjecting the biological sample to heat to remove the probe or a signal thereof.

22. The method of claim 21 , wherein the stripping agent is SDS and the probe is an antibody.

23. The method of claim 21 , wherein the probe is stripped by direct heat or microwave induced heat in the presence of a buffer.

24. The method of claim 18 , wherein removing the signal includes adding a charge transfer reagent to the biological sample, before step (ii), to quench residual fluorescence.

25. A method of probing multiple targets in a biological sample, comprising:

(a) optionally detecting hematoxylin and eosin staining of a biological sample containing multiple targets and stained with hematoxylin and eosin;

(b) optionally removing the hematoxylin and partially removing the eosin by washing the biological sample;

(c) subjecting the biological sample to an antigen retrieval process to expose one or more antigens in a region of interest;

(d) optionally applying a blocking reagent to block against nonspecific binding of one or more antigens;

(e) removing fluorescent hematoxylin and eosin signals from the biological sample;

(f) binding at least one probe to one or more targets present in the biological sample;

(g) detecting a signal from the at least one bound probe; and

(h) optionally removing the signal and optionally repeating steps (f) through (h);

wherein removing the fluorescent hematoxylin and eosin signals from the biological sample comprises:

(1) washing the biological sample using a buffered aqueous solution to remove the hematoxylin and partially remove the eosin; and contacting the biological sample with a charge transfer reagent to quench residual fluorescence;

(2) washing the biological sample using a buffered aqueous solution to remove the hematoxylin and partially remove the eosin; contacting the biological sample with hydrogen peroxide and a metal salt to generate hydroxyl radicals; and optionally irradiating the biological sample; or

(3) washing the biological sample using a buffered aqueous solution to remove the hematoxylin and partially remove the eosin; contacting the biological sample with an electron transfer reagent; and irradiating the biological sample to remove residual fluorescence.

26. The method of claim 25 , wherein removing the signal in step (h) comprises contacting the biological sample with a bleaching agent or stripping agent or subjecting the biological sample to heat to remove the at least one probe or a signal thereof.

27. The method of claim 25 , wherein said at least one probe is a protein binder.

28. A method of probing multiple targets in a biological sample, comprising:

(a) optionally detecting hematoxylin and eosin staining in a biological sample containing multiple targets and stained with hematoxylin and eosin;

(b) removing fluorescent hematoxylin and eosin signals from the biological sample;

(c) binding at least one probe to one or more targets present in the biological sample;

(d) detecting a signal from the at least one bound probe; and

(e) optionally removing the signal and optionally repeating steps (c) through (e);

wherein removing the fluorescent hematoxylin and eosin signals from the biological sample comprises:

(1) washing the biological sample using a buffered aqueous solution to remove the hematoxylin and partially remove the eosin; and contacting the biological sample with a charge transfer reagent to quench residual fluorescence;

(2) washing the biological sample using a buffered aqueous solution to remove the hematoxylin and partially remove the eosin; contacting the biological sample with hydrogen peroxide and a metal salt to generate hydroxyl radicals; and optionally irradiating the biological sample; or

(3) washing the biological sample using a buffered aqueous solution to remove the hematoxylin and partially remove the eosin; contacting the biological sample with an electron transfer reagent; and irradiating the biological sample to remove residual fluorescence.

29. The method of claim 28 , wherein removing the signal in step (e) comprises subjecting the biological sample to at least one of a bleaching agent, a protein denaturant, a DNA denaturant, heat, or SDS.

30. The method according to claim 28 , wherein the at least one probe is a FISH probe.