Microdissection fixation

For rodent species, including the rat and mouse, half of each litter is preserved for microdissection following fixation. The other half of each litter is subjected to a gross examination of... 

Key words Laser capture microdissection, Immunocytochemistry, Immunohistochemistry, Fixation,... 

This chapter summarizes our experience with this approach and describes some of the critical parameters involved in the preparation of rat neural tissue for LCM, with particular emphasis on protocols applied to the Arcturus Autopix LCM. This instrument has been superceded by more recent models, which are now marketed and distributed by MDS Analytical Technologies (http // www.moleculardevices.com). The principles described here, however, are applicable to tissue preparation for most LCM instruments. We describe two basic protocols for the isolation of samples of rat brain, first from unstained microdissected regions, and second, from brain cells that express specific antigens identified by immunostaining. In addition, we compare the effects of different fixation conditions on tissue recovery and RNA content using real-time QPCR. 

To allow efficient extraction of intact mRNA, it is recommended that alcohol fixatives be used in lieu of cross-linking agents such as paraformaldehyde. In our experience protocols that have been developed for studying paraformaldehyde-fixed paraffin-embedded archival specimens in tandem with RNA amplifications produce low yields of RNA from microdissected brain regions and picked cells. Fixation of tissue with methanol or ethanol preserves immu-noreactivity and mRNA in many cases. Accordingly, we immerse... 

Su JM, Perlaky L, Li XN, Leung HC, Antalffy B, Armstrong D, Lau CC (2004) Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues. Brain Pathol 14 175-182... 

Formalin-fixed tissue has extensive protein crosslinking because of the formation of methylene bridges during formalin fixation. The crosslinks limit the amount of protein that can be retrieved from a tissue section. In general, microdissected FFPE tissue is best suited to genomic analyses. 

Protocol 2.1 Restriction Enyzme Fragmentation of Probe DNA, 32 Protocol 2.2 Eabelinc with Terminal Deoxynucleotidyl Transferase, 33 Protocol 2.3 Microdissection of Polyeene Chromosomes for DOP-PCR, 34 Protocol 2.4 Primary DOP-PCR Amplification, 36 Protocol 2.5 Secondary DOP-PCR Amplification, 39 FIXATION METHODS, 41 Embryos, 41 Egg Chambers, 42 Other Tissues, 42... 

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