Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
2000-02-29
2003-11-18
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C435S005000, C435S007100, C435S007200, C435S007210, C435S007320, C435S007940, C435S007950, C435S040500, C436S503000, C436S518000
Reexamination Certificate
active
06649368
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Invention
The present invention generally relates to the enhancement of immunohistochemical staining of fixed tissue samples. More particularly, an embodiment of the invention relates to the enhancement of immunohistochemical staining of embedded formalin-fixed tissue samples using a single composition in a single step.
2. Description of Related Art
Tissue sections obtained from clinical or animal experimentation frequently have been fixed, embedded and stored in a form suitable for later examination by microscopy. Traditional fixation methods frequently have employed aldehyde fixatives, which fix the tissue by causing cross-linking reactions within and between tissue proteins. Cross-links tend to preserve tissue morphology and integrity, harden the tissue for slicing, and inhibit microbial attack. After tissue samples have been fixed, they are typically embedded in an embedding medium so that the samples may be cut into thin sections. Paraffin is the most common embedding medium, although acrylamide and celloidin may also be used.
Aldehyde fixation tends to cause substantial changes to the structure of the tissue sample. These changes often tend to cause the antigens that may be present in the tissue samples to lose their reactivity toward antibodies that target such antigens. One effect of formalin fixation is to substantially lock the three dimensional shape of protein molecules within the tissue samples. Because of the recent development of new immunohistochemical reagents, immunohistochemical analyses may now be performed that were impossible to perform at the time many tissues were originally stored. Therefore, a number of procedures have been developed which could reverse some of the changes produced by aldehyde fixation, and enhance the immunohistochemical staining properties of the tissue sample.
One method for improving the staining abilities of tissue samples which have been fixed in formalin and embedded in acrylamide gel relates to treatment of acrylamide gel embedded tissue in 1.0% 2-mercaptoethanol for 15 minutes, followed by rinsing with phosphate buffered saline. This treatment allowed the tissue samples to be stained by a number of staining reagents. A method for restoring the imnunohistochemical staining properties of tissue samples is described in U.S. Pat. No. 5,244,787 to Key et al. This method involved removing the embedding medium in a pretreatment step. For Paraffin-embedded tissue samples, this pretreatment was accomplished by e clearing the tissue samples in xylenes and rehydrating the samples. After the embedding medium has been removed, the sample may be heated in either de-ionized water, an aqueous solution of a zinc salt, or an aqueous solution of a lead salt. The tissue samples were reported to show improved immunohistochemical staining properties when heated in a microwave oven. Improvement was reportedly seen when the solution was heated to its boiling temperature. In general, microwave heating appears to have been found by Key et al to give better results than conventional heating. Solutions containing zinc or lead salts apparently gave significantly better results than de-ionized water.
Another method for restoring the immunohistochemical staining properties of tissue samples is described in U.S. Pat. No. 5,578,452 by Shi et al. In this method the formalin-fixed embedded tissues were treated with a solution of an aldehyde releasing reagent. The aldehyde releasing reagent may release aldehyde from the tissue sample by reacting with the aldehyde in a substantially irreversible manner to form a non-aldehyde derivative.
A number of investigators have investigated the importance of several reaction conditions with respect to enhancement of immunohistochemical staining ability. In general it has been found that the pH of the solution and temperature tend to have the most effect on the staining ability of the tissue samples. In general, with some limitations, the higher the temperature during the enhancement of the tissue samples, the better the staining enhancement tends to be. The effect of pH tends to be dependent on the type of antibody being used in the staining process and may be optimized for the antibodies to be used during the staining procedure.
The above mentioned methods inadequately address, among other things, the restoration of paraffin embedded tissue samples in a single reaction step. The procedures described above are usually performed on deparaffinized samples. Typically, the paraffin embedding medium is removed from the samples by successive immersion through a series of xylenes. Following removal of the paraffin embedding medium, the tissue must then be rehydrated by treatment with a series of ethanol-water solutions ranging typically from 100% ethanol to 90% ethanol. Finally, after the sample is rehydrated, the sample must be treated with a solution to reverse the effects of formalin fixation a step known as unmasking. It is therefore desirable that a single solution be provided that allows the steps of deparaffination (or de-embedding), rehydration, and unmasking of embedded tissue samples to be combined.
SUMMARY OF THE INVENTION
An embodiment of the invention relates to a liquid composition for enhancing the immunohistochemical staining ability of tissue samples. The composition preferably includes an aqueous solution of a removing agent and a tissue activating agent. The composition preferably substantially simultaneously: (i) removes the embedding medium from the tissue; (ii) improves immunohistochemical staining of the tissue in comparison to tissue that has not been contacted with the composition; and (iii) substantially hydrates the tissue. The removing agent is adapted to substantially remove an embedding medium from a tissue sample. The removing agent is preferably an emulsifier, more preferably a surfactant. The removing agent preferably includes one or more of an amphoteric, anionic, cationic, or nonionic surfactant. The tissue activating agent is adapted to alter the morphology of a component of the tissue sample. The tissue activating agent preferably includes a buffering agent or a metal salt. The pH of the composition is preferably adjusted to lie between 5 to 10 by addition of an acid or base.
In another embodiment the composition preferably includes an aqueous solution of SIMPLE GREEN. Simple Green has been described in U.S. Pat. No. 5,856,289 as: by weight about 5.8% ethylene glycol monobutyl ether, about 3.75% nonylphenol ethoxyate, about 1.5% tetrapotassium pyrophosphate and about 88.95% water. SIMPLE GREEN is a non-toxic, biodegradable, environmentally safe detergent concentrate which may provide a mixture of emulsifiers. The composition may include a buffering agent. The pH of the solution is preferably adjusted to lie between 5 and 10 by addition of an acid or a base. The composition preferably substantially simultaneously: (i) removes the embedding medium from the tissue; (ii) improves immunohistochemical staining of the tissue in comparison to tissue that has not been contacted with the composition; (iii) substantially hydrates the tissue.
In another embodiment of the composition the removing agent includes aqueous solutions of at least one of the following emulsifiers, including detergents and surfactants: Igepal-630 (sigma #3021); Tween 20 (sigma #P7949); Brij 35 (sigma #P 1254); Brij 90 (sigma #P 1254); Triton X-100 (sigma #T9284); CD TAB (sigma #C5335); and Tween 80 (sigma #P8074). Reference to “sigma”is reference to ©1999 Sigma—Aldrich catalog entitled: “Biochemicals and Reagents for Life Science Research”, which is incorporated herein by reference.
The composition is preferably used to enhance the immunohistochemical staining of the tissue sample. In a preferred method the tissue samples are cut into sections of less than 5 microns. The tissue samples may be mounted on a positively charged slide. The sections are preferably dried at about 58° C. for one hour. After this time the samples are preferably submersed within the compos
Aghassi Nora Betyousef
Ardi Paul John
Franceschini Kim
Cell Marque Corporation
Chin Christopher L.
Grun James L.
Jackson & Walker, LLP
LandOfFree
Composition and method for treating tissue samples does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Composition and method for treating tissue samples, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Composition and method for treating tissue samples will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3166622