Chemistry: analytical and immunological testing – Including sample preparation
Patent
1988-07-29
1991-12-17
Warden, Robert J.
Chemistry: analytical and immunological testing
Including sample preparation
422 99, 422100, 422101, G01N 100, B01L 302
Patent
active
050735047
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND
The present invention relates in general to apparatus and methods for applying reagents to and removing reagents from slides. In particular, the present invention relates to apparatus and methods for immunohistochemical staining of biological material on glass slides using economically small amounts of reagents.
Histology refers to the study of the microscopic structure of cells, tissues, and organs. The traditional method of analyzing the microscopic structure of an organ is to cut extremely thin sections of tissue on a special device called a microtome. After embedding the tissue in paraffin, a microtome can cut it 3-5 microns thick. Next, the section of tissue is attached to a glass slide. The thin tissue section is then stained with dyes or chemicals which are chosen for their ability to enhance various features in the tissue specimen.
Different dyes may be used to highlight different structures of cells or organs. For example, hematoxylin is a dye which stains cell nuclei purple, and eosin, often used in combination with hematoxylin, is a chemical compound which stains the cell cytoplasm red.
The staining process typically involves dipping the slides into small vats or jars containing the chemicals and/or dyes. Each vat or jar typically contains 0.2-2.0 liters.
More recently, a new type of staining process using specific antibody or DNA probes has been developed. These procedures have the advantage of high specificity and sensitivity, and are commonly used as an adjunct to traditional staining procedures. However, because the probes are far more expensive than dyes or stains, the probes are applied by a special technique which uses only several drops (about 50-200 microliters) of reagent.
In a common procedure for antibody-mediated staining ("immunohistochemistry"), frozen or paraffin-treated thin tissue sections (about 3-5 microns in thickness) are placed on glass slides. Frozen tissue sections are placed in acetone for temporary storage and are washed in buffered saline before use. Paraffin-treated tissue sections are baked at 56.degree. C., deparaffinized in xylene, rehydrated in decreasing grades of ethanol and treated for 30 minutes with dilute hydrogen peroxide. The tissue sections are washed with buffered saline before use.
Next, each slide is blotted dry without disturbing the thin tissue section. Each slide is then placed in a humidified box, tissue side up. An appropriate first reagent is pipetted onto the tissue in a volume of 50-200 microliters. After the reagent has been added to each slide, the humidified chamber is closed, lest the reagent evaporate and leave the tissue dried and, therefore, spoiled.
After the appropriate incubation period (usually 30-60 minutes) each slide is removed from the humidified box and placed in a slide rack. The rack is immersed in buffered saline which is periodically replaced. After washing sufficiently to ensure that the first reagent is removed, each slide is manually removed from the saline, carefully dried and placed into the humidified chamber. A second reagent is then pipetted onto each slide and the slides are again incubated for 30 minutes. This procedure is repeated until all the incubation steps have been completed (usually 3-4 times).
A severe drawback of the present technique is that it is labor intensive. Steps which require individual manual handling of the slides (such as drying and transferring steps) are tedious and time consuming. Handling each slide at each incubation step (transferring a slide from the slide rack to the humidified box, drying the slide, and replacing the slide back in the rack) requires approximately one minute. For a typical run of 50 slides with four incubation steps each, this translates into 3.3 hours of technician time in addition to the time required for incubation and washing (about 45-75 minutes for each step).
An additional problem with the present technique is that a relatively large inequality of incubation time occurs when about 20 or more slides are stained. Because a significant amount of time i
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Santiago Amalia L.
Warden Robert J.
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