US 20110229879A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0229879 A1 CHURUKIAN (43) Pub. Date: Sep. 22, 2011 (54) METHODS AND COMPOSITIONS FOR Publication Classification NUCLEAR STANING (51) Int. Cl. (75) Inventor: Charles J. CHURUKIAN, CI2O I/68 (2006.01) Rochester, NY (US) (52) U.S. Cl. ......................................................... 435/6.1 (73) Assignee: UNIVERSITY OF ROCHESTER, Rochester, NY (US) (57) ABSTRACT The present invention relates to compositions, methods, and (21) Appl. No.: 13/052,791 kits Suitable for detecting nucleic acids inabiological sample. The nuclear staining composition of the present invention (22) Filed: Mar. 21, 2011 contains a pH buffering reagent, a solubilizing reagent, a basic dye, and an aqueous medium. The composition can be Related U.S. Application Data used alone to detect nucleic acids in a biological sample or in (60) Provisional application No. 61/315,483, filed on Mar. combination with other histological dyes for nuclear counter 19, 2010. staining. Reticulin Stain Patent Application Publication Sep. 22, 2011 Sheet 1 of 3 US 2011/0229879 A1 Reticulin Stain Figures 1A-1B Patent Application Publication Sep. 22, 2011 Sheet 2 of 3 US 2011/0229879 A1 Iron Stain Figures 2A-2B Patent Application Publication Sep. 22, 2011 Sheet 3 of 3 US 2011/0229879 A1 Alcian Blue Figures 3A-3B US 2011/0229.879 A1 Sep. 22, 2011 METHODS AND COMPOSITIONS FOR composition of the present invention does not precipitate in NUCLEAR STANING Solution and has a shelf-life of at least one year. In addition, nuclear staining with the composition of the present invention achieves brighter, more brilliant nuclear staining showing 0001. This application claims the benefit of U.S. Provi Superior tissue architecture and cellular detail within seconds sional Patent Application Ser. No. 61/315,483, filed Mar. 19, of exposure. Finally, unlike conventional dyes that can 2010, which is hereby incorporated by reference in its weaken within weeks of staining, the nuclear staining com entirety. position of the present invention is extremely lightfast, with no significant fading observed over time. FIELD OF THE INVENTION 0002 The present invention relates to compositions and BRIEF DESCRIPTION OF THE DRAWINGS methods suitable for detecting nuclear elements in a biologi cal sample. 0010 FIGS. 1A-1B are light microscopy images of reticu lum fiber staining in liver tissue sections using Gomori's reticulum staining procedure. Following staining for the BACKGROUND OF THE INVENTION reticulum, nuclear counterstaining was carried out using 0003. Histochemical procedures performed on surgical, nuclear fast red solution (FIG. 1A) for five minutes and the autopsy, and biopsy tissue and cell Samples for diagnostic and strong fast red solution (0.1% pararosaniline solution) of the research purposes generally involve the use of a nuclear coun present invention (FIG. 1B) for 10 seconds. terstainto delineate tissue architecture and cellular detail. The 0011 FIGS. 2A-2B are light microscopy images of liver nuclear counterstain is usually a dark color to contrast a sections stained for iron using the Perl’s ferric iron method. lighter dye used to label the cytoplasmic or extracellular Following the staining procedure for the detection of iron, structures of interest. nuclear counterstaining was carried out using nuclear fast red 0004. A nuclear counterstain is the desired stain for a solution (FIG. 2A) for 5 minutes and the strong fast red number of histological staining procedures including, for solution of the present invention (FIG. 2B) for 10 seconds. example, Gomori's reticulum, Pearl's ferric iron, Alcian blue 0012 FIGS. 3A-3B are light microscopy images of small for acidic mucins, Jones basement membrane, Churukian's bowel sections stained with alcian blue for acidic mucins. ammonical silver for melanin, melanin bleach, and Lillie's Following staining with alcian blue, nuclear counterstaining ferrous iron uptake method for melanin. While nuclear fast was carried out using nuclear fast red solution (FIG. 3A) for red is most commonly used in methods requiring ared nuclear 5 minutes and the strong fast red solution of the present counterstain, this dye involves incubations periods of 5-10 invention (FIG. 3B) for 20 seconds. minutes, and often fades within just weeks of staining. In addition, nuclear fast red is not compatible with all of the above noted Staining procedures and has limited Stability in DETAILED DESCRIPTION OF THE INVENTION Solution. Accordingly, there is a need in the art for a nuclear 0013. A first aspect of the present invention relates to a dye that requires shorter incubation times, has increased com composition for detecting nucleic acids in a biological patibility with other staining procedures, is resistant to fad sample. The composition contains a pH buffering reagent that ing, and has long term stability. These needs are particularly, maintains the composition at a pH of less than 5. The com though certainly not exclusively, applicable for red nuclear position further contains a solubilizing reagent, a basic dye, dyes. and an aqueous medium. 0005. The present invention is directed to overcoming 0014. In accordance with this aspect of the present inven these and other deficiencies in the art. tion, the composition contains a pH buffering reagent that maintains the composition at a pH of less than 5. More pref SUMMARY OF THE INVENTION erably, the pH buffering reagent maintains the composition at 0006. A first aspect of the present invention relates to a a pH of less than 3. Even more preferably, the pH buffering composition for detecting nucleic acids in a biological reagent maintains the compositionata pH of between 2.3-2.7. sample. This composition contains a pH buffering reagent 0015. Any weak acid or weakly acidic buffer can be used that maintains the composition at a pH of less than 5. The as a pH buffering reagent in the composition of the present composition further contains a solubilizing reagent, a basic invention. Suitable pH buffering reagents include, but are in dye, and an aqueous medium. no way limited to lactic acid, acetic acid, citrate acid, oxalic 0007. A second aspect of the present invention relates to a acid, formic acid, hydrochloric acid, acetate buffer, citric method of detecting nucleic acids in a biological sample that acid/disodium phosphate buffer, sulfuric acid/sodium phos involves exposing the biological sample to a composition of phate buffer, malonic acid/sodium hydroxide buffer, sodium the present invention under conditions effective to label the acetate/acetic acid buffer, acid phosphate/hydrochloric acid nucleic acids in the biological sample. The method further buffer, and hydrochloric acid/sodium citrate buffer. The pH of involves viewing the biological sample using light micros the buffering reagent is preferably about 2.5, although buffers copy to detect the nucleic acids in the biological sample. having a pH of about 2.3 to about 2.7 are also suitable for use. 0008. A third aspect of the present invention relates to a kit In one embodiment of the present invention, the composition containing the composition of present invention and instruc contains lactic acid as the pH buffering reagent at a concen tions for using the composition for detecting nucleic acids in tration of about 0.5% to about 0.7% by volume. Ideally, the a biological sample. lactic acid concentration of the composition of the present 0009. As described herein, the composition of the present invention is at about 0.6% by volume. In an alternative invention offers many advantages over currently available embodiment of the present invention, the composition con nuclear dye Solutions such as nuclear fast red. Firstly, the tains acetic acid as the pH buffering reagentata concentration US 2011/0229.879 A1 Sep. 22, 2011 of about 1% to about 5% by volume. Ideally, the acetic acid about 0.2% by weight. The dye can also be present in an concentration of the composition of the present invention is amount of about 0.04% to about 0.18% by weight or 0.05% to about 3% by volume. 0.15% by weight. 0016. The composition of the present invention also con 0019. In one embodiment of the present invention, the tains a solubilizing reagent. Suitable solubilizing reagents composition contains pararosanilin at a concentration of include low molecular weight emulsifiers or Surfactants, about 0.05% to about 0.15% by weight. More ideally, the including PEG-ylated sorbitan fatty acid esters, such as concentration of pararosanilin in the composition of the polysorbate 20 (Tween 20R). Alternatively, the solubilizing present invention is 0.1% by weight. reagent can be a non-ionic Surfactant having a hydrophilic polyethylene oxide group such as TritonX-100R). The con 0020. The composition of the present invention further centration of the Solubilizing reagent in the composition of contains an aqueous medium, Such as, for example, deionized the present invention is about 0.008% to about 0.05%. More Water. preferably, the concentration of the solubilizing reagent in the 0021. The composition of the present invention has a pH composition of the present invention is about 0.01% to about of about 2 to about 3. More preferably, the pH of the compo 0.04%. In one embodiment of the present invention, the com sition is between about 2.4 to about 2.6. Most preferably, the position contains polysorbate 20 at a concentration of about pH of the composition of the present invention is about 2.5. 0.01% to about 0.04% by volume. More ideally, the polysor 0022. A significant benefit of the composition of the bate 20 concentration of the composition of the present inven present invention is its stability over time. Unlike other com tion is about 0.025% by volume. In an alternative embodi ment of the present invention, the composition contains monly used dyes, such as nuclear fast red which has a limited TritonX-100R at a concentration of about 0.01% to about shelf-life of about four months because of precipitation, the 0.04% by volume.