Manual Microdissection 显微解剖
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Manual Microdissection
This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study.
Method
The approach of the NCI Prostate Group is to dissect on a standard inverted microscope using a 30 gauge needle on a syringe as the microdissecting tool.
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While viewing the tissue through the microscope, the cell population of interest should be gently scraped with the needle. The dissected cells will become detached from the slide and form small dark clumps of tissue that can be collected on the needle by electrostatic attraction. Several small tissue fragments can be procured simultaneously. Collection of an initial fragment on the tip of the needle will assist in procuring subsequent tissue. The tip of the needle with the procured tissue fragments should be carefully placed into a small PCR tube containing the appropriate buffer. Gentle shaking of the tube will ensure the tissue detaches from the tip of the needle.
- Placement of frozen tissue sections directly on agarose coated slides can be helpful in maintaining enzyme stability. Additionally, the agarose gels can be prepared or soaked in custom buffers that will bathe the frozen section prior to and during the microdissection, e.g., pH, salt concentration, proteinase inhibitors, etc., can be varied specifically for a given enzyme. Some members of the NCI Group also prefer to use the agarose coated slide microdissection approach for recovery of mRNA. Slides for microdissection are prepared by placing 200 µl of warm agarose on standard uncoated glass slides, covering with a glass slip, and allowing the gel to polymerize. The glass slip is removed from the slide and the frozen tissue section is immediately placed onto the agarose gel. The freshly cut section should be transferred directly from the cryostat to the agarose coated slide.
References:
Emmert-Buck, MR, Roth, MJ, Zhuang, Z, Campo, E, Rozhin, J, Sloane BF, Liotta, LA, and Stetler-Stevenson, WG. Increased gelatinase A (MMP-2) and cathepsin B activity in invasive tumor regions of human colon cancer samples. Am. J. Pathol 145(6):1285-90, 1994.
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