Fresh tissue microdissection

A minimum of 50% of fetuses are to be examined for visceral alterations and a minimum of 50% for skeletal abnormalities. When a fresh tissue microdissection technique is being used for the visceral examination of rabbit fetuses, all fetuses should be examined for both visceral and skeletal abnormalities. 

Key words Fetal examination, Visceral, Fresh soft tissue. Fixed tissue. Microdissection... 

Jin L, Majerus J, Oliveira A, et al. Detection of fusion gene transcripts in fresh-frozen and formalin-fixed paraffin-embedded tissue sections of soft-tissue sarcomas after laser capture microdissection and RT-PCR. Diagn. Mol. Pathol. 2003 12 224-230. 

The sample materials from which proteins for proteomics studies may be extracted include fresh or snap-frozen cells from varied sources such as biological fluids, (serum, urine, plasma) and solid tissues such as biopsy specimens. Moreover, proteins isolated from ethanol-fixed paraffin-embedded tissues can be utilized for MS analysis.2 Protocols for the identification of proteins from formalin-fixed paraffin-embedded (FFPE) tissues have been recently developed.3 4 FFPE materials are the most common forms of biopsy archives utilized worldwide, and represent an important advancement for the large-scale interrogation of proteins in archival patient-derived materials. Finally, laser capture microdissected tissues have been successfully used for MS analysis.45... 

The remaining fetuses are submitted to fresh soft tissue examination or preserved for fixed visceral examination (see Note 2). Various techniques are available typically based on Wilson s sections of the head and body or Wilson s sections of the head only with microdissection of the body. The techniques used for the preparation and examination of the fetuses are described in Chapters 16-21. 

This chapter describes methods for the examination of fetal abdominal and thoracic soft tissues by microdissection on either fresh (non-rodent) or fixed (rodent) specimens in order to detect structural abnormalities. With hundreds of fetuses examined for each species (rodent and non-rodent) in regulatory reproductive toxicity assessments (ICH, http //www.ich.org/fileadmin/Public Web Site/ICH ... 

Following a gross external examination, all fetuses of non-rodent species, including the rabbit and minipig, are submitted to a fresh soft tissue examination by microdissection, after removal of the heads from half of the litter for serial sectioning. 

The aim of the examinations is to reveal any differences with respect to the expected position, structure, size, and shape of the abdominal and thoracic organs/tissues. Both fresh and fixed microdissection methods allow subsequent histopatho-logical examination of the tissues if considered appropriate. 

Microdissection allows each non-rodent fetus to be submitted to both soft tissue and skeletal examinations (at least of the body). Similarly, a simpler gross examination of fresh soft tissues can be performed on the abdominal and thoracic organs of rodent fetuses destined for skeletal examination (see Note 1). 

A common issue with microdissected tissue is minimal cell number on any given LCM cap. To effectively increase the cell number and protein concentration for a volume of lysis buffer, it may be necessary to solubilize 2-3 LCM caps in 1 vol of lysis buffer. This is done by sequentially solubilizing the cells on LCM caps in the same volume of lysis buffer. To illustrate the process, one LCM cap is solubilized in lysis buffer. This first cap is discarded and a fresh LCM cap containing microdissected material is placed on a tube containing the lysis buffer used previously to solubilize the first LCM cap. The cells on this second cap are solubilized in the lysis buffer, effectively increasing the protein concentration in a given volume of buffer. 

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