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Page 2 of 2 |
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| The Tissue Bank Laboratories | |||
Fig 5. The tissue processing machine
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Tissue Processing and EmbeddingTissue blocks taken from eighteen sampling regions during the fixed dissection must be processed before the tissue can be stained for analysis. This is done using the processing machine pictured to the left. Briefly, the blocks (still in their processing cassettes) are placed in a metal cage, which is then put in the chamber. The machine then floods the chamber with a series of liquids, according to the program selected. (For routine processing, a 24-hour program is used.) The final step of the procedure is to infiltrate the tissue blocks with wax, ready for embedding. |
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When the processor has finished, the blocks are placed in embedding troughs, which are then filled with molten wax. Labelled embedding cassettes are placed over the top, and the wax is topped up before being allowed to set. Processing and embedding preserves the morphology of the tissue and makes the anatomy easier to see when the tissue is stained. |
Fig 6. The embedding machine
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Sectioning TissueSo, by this stage, the tissue for analysis has been fixed in formalin, infiltrated with paraffin wax and embedded in a wax block. The next step is to cut extremely thin sections of tissue for staining. This is done using the equipment pictured to the right. |
Fig 7. The microtome (foreground)
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Tissue sections are cut on a microtome and then floated out on a water bath. Once the sections have flattened out, they are collected onto slides and allowed to dry. The sections are then ready for staining with a variety of different dyes and/or antibodies. |
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HistologyThere are two main types of staining techniques employed by the Tissue Bank for diagnostic work. The first uses dyes which bind to a particular class of structures within the tissue. For example, haematoxylin stains nuclei blue, while eosin colours cytoplasmic constituents pink and red blood cells a glassy orange-red. These two dyes are commonly used together in a routine diagnostic stain (referred to as haematoxylin and eosin, or H&E). Eosin binds to Lewy bodies, one of the main pathological features of Parkinson's disease. See Fig 4 for an example of H&E staining in normal (a) and PD (b) substantia nigra. The dark brown substance is neuromelanin, a substance found in dopaminergic neurons. These are the neurons that degenerate in PD. The second technique is called immunohistochemistry, and makes use of antibodies that bind to specific molecules. The Tissue Bank routinely uses antibodies against alpha-synuclein, ubiquitin, tau and beta-amyloid. The first two are the main components of Lewy Bodies, while the second two are indicative of Alzheimer's disease pathology. When sections have been cut, all regions are stained with H&E, and two with Luxol fast blue/periodic acid Schiff (LFB/PAS). (LFB is a myelin stain, while PAS shows up carbohydrates and other substances as a deep magenta colour.) The stained slides are examined by a neuropathologist to assess the quality of the tissue, and to determine whether analysis of the pathology will require anything more than the standard screen. Following the primary screen, the next step is to carry out the immunohistochemical staining, plus any other techniques the neuropathologist thinks necessary. The standard secondary screen involves staining all regions for alpha-synuclein and selected regions for tau, beta-amyloid and ubiquitin.
When all the work for a case has been completed, the case is passed on to the neuropathologists for diagnosis.
The aim of the Tissue Bank is to supply tissue to researchers investigating Parkinson's disease. We have received requests for fresh-frozen, fixed-frozen and paraffin-embedded tissue. First, the clinical and neuropathological information is used to select cases with the appropriate profile. Then, the relevant type and amount of tissue is retrieved as described below. Paraffin-embedded sections are cut from the diagnostic blocks using the microtome, as described above. For frozen sections or blocks, the region of interest must first be identified using the photographs taken at the dissection. Once the relevant blocks have been retrieved, sections are cut on a cryostat. (A cryostat is simply a special type of microtome contained within a refrigerated unit.) Tissue blocks or sections are transported to the requesting scientist by courier, using packaging appropriate for the tissue type. |
Fig 8. Sections of substantia nigra stained with H&E
b: with PD (right)
Fig 9. Examining stained sections under the microscope |
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This concludes the tour of the laboratory side of the Tissue Bank operation. (Page 2 of 2) |
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a: without PD (left)
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