Methods of analyzing an H and E stained biological sample

1. A sequential method of preparing a hematoxylin and eosin stained biological sample for analysis comprising:

(a) providing a hematoxylin and eosin stained biological sample containing multiple targets;

(b) observing the hematoxylin and eosin si al from the stained biological sample;

(c) removing the hematoxylin and partially removing the eosin by washing the sample;

(d) contacting the sample with an electron transfer reagent where the electron transfer reagent is a borate salt represented by the following structural formula:

wherein:

each R1, R2, and R3 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,

R4 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;

(e) irradiating the sample of step (d) to remove residual fluorescence.

2. The method of claim 1 , wherein irradiating the sample in step (e) is accomplished by exposing the sample to light of 350 nm-1.3 μM in wavelength.

3. The method of claim 2 , wherein irradiating the sample in step (e) is accomplished by exposing the sample to light of 400-700 nm in wavelength.

4. The method of claim 1 , wherein irradiating the sample in (e) is carried out in the presence of a buffer at pH of 5-9.

5. The method of claim 1 wherein step (e) is carried out at the temperature of 4-50° C.

6. The method of claim 1 , wherein step (e) is performed for about 20 seconds to about 15 minutes.

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

8. The method of claim 7 wherein each R 1 , R 2 , and R 3 is phenyl and R 4 is butyl, or benzyl.

9. The method of claim 1 wherein M + is an inorganic cation selected from the group consisting of Li + , Na + or K + .

10. The method of claim 1 further comprising the steps of:

(f) binding at least one probe capable of signal generation to one or more targets to the sample of step (e);

(g) observing a signal from the probe bound in step (f); and

(h) optionally contacting the sample with a bleaching agent and repeating steps (f) through (g).

11. The method of claim 10 wherein the bleaching agent is a photoactivated chemical bleaching agent and the step (h) further comprises irradiating the sample by exposing the sample to light of 350 nm-1.3 μM in wavelength.

12. The method of claim 11 , wherein irradiating the sample is accomplished by exposing the sample to light of 400-700 nm in wavelength.

13. The method of claim 10 , wherein the probe in step (f) comprises a fluorescent signal generator, and the signal observed in step (g) is a fluorescent signal.

14. The method of claim 10 , wherein steps (f)-(h) are repeated two or more times.

15. The method of claim 10 , further comprising measuring one or more intensity values of the signal observed in observing step (g).

16. The method of claim 15 further comprising correlating the intensity value with an amount of target present in the sample.

17. The method of claim 10 , wherein the electron transfer agent in step (d) is the same as the bleaching agent in step (h) and step (h) further comprises irradiating the sample by exposing the sample to light of 350 nm-1.3 μM in wavelength.

18. A sequential method of probing multiple targets in a hematoxylin and eosin stained biological sample comprising:

(a) providing a hematoxylin and eosin stained biological sample containing multiple targets;

(b) observing the hematoxylin and eosin signal from the stained biological sample;

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

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

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

(f) contacting the sample with an electron transfer agent reagent where the electron transfer reagent is a borate salt represented by the following structural formula:

wherein:

each R1, R2, and R3 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,

R4 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;

(g) irradiating the sample of step (f);

(h) binding at least one probe capable of signal generation to one or more targets present in the sample of step (g);

(i) observing a signal from the probe bound in step (h); and

(j) optionally contacting the sample with a bleaching agent and repeating steps (h) through (i).

19. The method of claim 18 wherein step (h) wherein the probe is a protein stain.

20. The method of claim 18 wherein the bleaching agent is a photoactivated chemical bleaching agent and the step (j) further comprises irradiating the sample by exposing the sample to light of 350 nm-1.3 μM in wavelength.

21. The method of claim 18 , wherein each R 1 , R 2 , and R 3 is an independently optionally substituted aryl and R 4 is an optionally substituted alkyl.

22. The method of claim 21 , wherein each R 1 , R 2 , and R 3 is phenyl and R 4 is butyl or benzyl.

23. The method of claim 18 wherein M + is an inorganic cation selected from the group consisting of Li + , Na + or K + .

24. The method of claim 18 wherein the electron transfer agent in step (f) is the same as the bleaching agent in step (j) and step (j) further comprises irradiating the sample by exposing the sample to light of 350 nm-1.3 μM in wavelength.

25. A sequential method of probing multiple targets in a hematoxylin and eosin stained biological sample comprising:

(a) providing a hematoxylin and eosin stained biological sample containing multiple targets;

(b) observing the hematoxylin and eosin signal from the stained biological sample;

(c) removing the hematoxylin and partially removing the eosin by washing the sample;

(d) contacting the sample with an electron transfer agent reagent where the electron transfer reagent is a borate salt represented by the following structural formula:

wherein:

each R1, R2, and R3 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,

R4 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;

(e) irradiating the sample of step (d) to remove the residual eosin fluorescence

(f) binding at least one probe capable of signal generation to one or more targets present in the sample of step (e);

(g) observing a signal from the probe bound in step (f); and

(h) optionally subjecting the sample to a bleaching agent, DNA denaturant, heat, or a combination thereof and repeating steps (f) through (h).

26. The method of claim 25 , wherein each R 1 , R 2 , and R 3 is each independently an optionally substituted aryl and R 4 is an optionally substituted alkyl.

27. The method of claim 26 , wherein each R 1 , R 2 , and R 3 is phenyl and R 4 is butyl or benzyl.

28. The method of claim 25 wherein M + is an inorganic cation selected from the group consisting of Li + , Na + or K + .

29. The method of claim 25 wherein the electron transfer agent in step (d) is the same as the bleaching agent in step (h) and step (h) further comprises irradiating the sample by exposing the sample to light of 350 nm-1.3 μM in wavelength.

30. A method according to claim 27 wherein the at least one probe is a FISH probe.