Methods of analyzing an H and E stained biological sample

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).

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).