Microdissection staining

Numerous articles have demonstrated the availability of RNA extracted from a few cells or even a single cell taken by laser captured microdissection (LCM) system from archival FFPE tissue sections that had been previously stained by IFIC. Using a combination of pre-immunostained FFPE tissue section with LCM, a sensitive real-time quantitative RT-PCR can be achieved based on a few immuno-detected cells, creating a way to study pathophysiological gene regulation in a cell-specific manner in archival tissues housed in... 

Alcock, H. E., Stephenson, T. J., Royds, J. A., and Hammond, D. W. 1999. A simple method for PCR based analyses of immunohistochemically stained, microdissected, formalin-fixed, paraffin wax-embedded material. Mol. Pathol. 52 160-163. 

Mouledous L, Hunt S, Harcourt R, Harry J, Williams KL, Gutstein HB. Navigated laser capture microdissection as an alternative to direct histological staining for proteomic analysis of brain samples. Proteomics 2003 3(5) 610-615. 

Each paraffin-embedded section is collected on microscope slides and first examined under a microscope (lOx) to ensure that it contained sufficient tumor material and to eliminate possible contaminating normal tissues. Tumor and tumor-free areas are identified within 15 pm-thick deparaffinized sections lightly counterstained with hematoxylin and microdissected by gentle scraping with sterile scalpels into 1.5 ml polypropylene vials, using a hematoxylin and eosin-stained step section from the same block [4-7]. 

Do not allow the tissue section to dry on the slide at room temperature. Place the slide directly on dry ice or keep the slide in the cryostat at -20°C or colder until the slides can be either stained and microdissected, or stored at -80°C (see Note 6). 

Allow the stained slide to air dry briefly. Proceed directly with microdissection. The stained slide should not be re-frozen (see Note 11). 

Protease and/or phosphatase inhibitors may be added to compatible staining solutions (17). Complete Protease Inhibitor Cocktail tablets are water soluble. For protease inhibitor addition to the 70% ethanol solution, dissolve the tablet in 15 ml of dH20, and then add 35 ml ethanol. Limiting the time from staining to completion of microdissection also ensures preservation of cellular constituents. Frozen section samples for RNA and protein analysis should be stained and microdissected within 1 h. 

The limited sample material from microdissected biopsy lysates precludes the use of spectrophotometeric analysis of total protein prior to microarray printing. A microarray slide stained for total protein serves as a tool for normalizing spot intensity between samples (see Note 15). 

Microdissection via the laser adhesive technique may be a solution to this plight (Banks et al. 1999). The tissue sample (e.g., the punctate) is cut into slices with the microtome and the slices are stained with hematoxylin and eosin. You lay the stained cut on a glass plate and push it under an inverted microscope. Now you identify the tumor and lay a little tube on the interesting area. The bottom of the little tube is sealed with a UVA polymer film. The film thus touches the tissue. Now the cancerous parts of the tissue are glued to the film. This is done with a laser beam directed at the desired areas. Once the adhesion is complete, you take the little tube off, with film and the tissue that is stuck to it. The rest of the cut remains on the glass plate. Now the little tube is put on an Eppendorf cup like a lid. In the cup there is sample buffer. If you turn the cup aroimd, the sample buffer loosens the protein from the film (Figure 7.3). 

Bolster, J., Dithmar, H., Bmgemeister, R., Friedemann, G. Feucht, W. (2006). Flavonoids in plant nuclei detection by laser microdissection and pressure catapulting (LMPC), in vivo staining, and UV-visible spectroscopic titration. Physiologia Plantarum, 128, 163-174. 

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