|TISSUES >> PROCESSING AND CONSERVATION|
The preparation and conservation of tissues in a collecting center must be subject to standarized protocols. Is the function of the tissue bank to determine the different procedures in accordance with optimized protocols and normalize its application, independently of the sample origin.
The interesting organs are extracted and the different areas are dissected and processed in order to obtain fixed and frozen fresh material. This technique allows to preserve tissue for morphological and biochemical studies.
The processing of the tissue includes different stages as it is shown in the processing general scheme of the tissues..
With the purpose of research, the tissue obtained by necropsy must be manipulated and processed in appropriate conditions. Some factors should be taken on consideration: the post-morten interval, the processing (dissection, freezing, fixation...), the storage and transport conditions.
Ideally, the tissue is obtained immediately after the death of the animal. However in the practice, many times the extraction of the tissue is not possible until several hors later. The elapsed time is variable depending on the animal origin, but the time is tried to be minimized in order to carry out biochemical studies (genomics, proteomics...) apart from histological studies (histochemistry, inmunohistichemistry...) with the collected material. Not only the elapsed time is a variable to consider but also the conservation conditions of the cadaver until the moment of the necropsy. Finally, although the tissue extraction is a rapid procedure, is necessary to take into account the moving time that is variable depending on the animal origin.
Tissue extraction and area dissecting
The first phase consists in the extraction of the tissue. In the case of the brain, firstly the cranium is opened and the meninges eliminated. After the brain extraction, in the base of the cranium, the hypophysis and the trigeminum are extracted. ( see tissue extraction and areas dissection scheme ).
Once the brain has been extracted, it is sagitally divided in order to separate both hemispheres. The left hemisphere is submerged in formaldehyde for 5-15 days (depending on the specie and the brain size), it is cutted in coronal sheets and the different areas are dissected (in function of the cutted level) that are processed to be embedded in paraffin in order to make tissue blocks. The right hemisphere is also cutted in coronal sheets and the different areas are dissected and frozen rapidly and conserved at –80ºC. In some cases, interesting areas are fixed with paraformaldehyde, cryoprotected in sucrose and frozen. The cerebellum and brainstem are also dissected in sheets and some areas are selected to be fixed or frozen.
The selected areas of the different coronal cut levels, are the represented in the processing general scheme of the central nervous system and areas dissection..
In detail, the different anathomical regions of the brain are illustrated in the following images:
-Dorsal and ventral macroscopical view
- Macroscopical view of a set of coronal cuts.
In the case of require an specific dissection protocol different from the systematic stabled, the BTAC wild be able to modify the dissecting procedure in accordance with the requester necessities.
Sample processing and conservation
The tissue samples are processed and conserved according as three different specifications:
• Frozen fresh tissue at -80ºC: suitable for biochemical studies (genomics, proteomics...), in situ hybridization and, some histochemical and inmunohistochemical techniques after fixation of the sections.
• Fixed tissue in 10% of buffered formaldehyde and embedded in paraffin. (Paraffin embedding scheme): suitable for the routine histological studies, special histochemical staining, histochemistry and inmunohistochemistry.
• Fixed tissue in 4% buffered paraformaldehyde, cryoprotected in 30% of sucrose and frozen at -80ºC: applicable only in some cases. Suitable for histochemical and inmunohistochemical techniques and in situ hybridization.
The frozen fresh material is stored properly identified in –80ºC freezers, and the material included in paraffin is stored in specially designed cupboards.
The previously described methodology must be considered as the
standard procedure of permanent and systematic collection of samples.
If for the performing of a certain project is necessary an other kind
of processing, the BTAC will be able to prepare together with the main
researcher of the project, a different collection, processing and
conservation of the tissue according to the research purposes. Once the
number of cases required for the requester have been satisfied, the
BTAC would leave off its participation in the mentioned research
The material is remitted to the bank in different manners: