engleski

Preparation of mouse brain samples for electron microscopy

The morphological analysis represents the basic method used to determine the phenotype changes of the genetically modified mouse. In this way the observed phenotype changes are correlated to the genetic modification and enable the insight in the function of the investigated gene. The challenge of the morphological analysis increases with the increased complexity of the investigated tissues and organs. Therefore the most complex organ to be studied is the brain. In order to compare the brain neuroanatomy of the mutant and wild type mice, the brain could be studied at the gross-morphology level as a whole, at the light microscope level on histological sections, or at the ultrastructural level by electron microscopy (1). In order to compare the brain morphology at the ultrastructural level of the mutant and the wild type mouse, we used the mouse mutant Stam2Gt1Gaj, where due to the gene trap modification the function of Stam2 was hindered. Due to the brain size and the nature of electron microscopy analyisis only two brain regions were selected to be viewed by electron microscope, CA2 region of the hippocampus and the adjacent cerebral cortex. In order to compare the equivalent regions of the brain in the mutated and wild type mice the regions of interest were determined on vibratome sections, and the selected pieces of tissues were processed for electron microscopy. Adult, 3 months old mice were anesthetized with 1% avertin and perfusion fixation was performed. 0.1 M phosphate buffer was injected within the left heart ventricle in order to rinse the blood from the circulatory system. Afterwards, the animals were perfused with 12.5% glutaraldehyde, which resulted with initial fixation of the brain. Skull was opened with sharp scissors and the brain was removed and immersed in 6% glutaraldehyde for 1 hour. The brain was afterwards cut in 200 µ m thick sagital sections using vibratome. After determination of the brain regions of interest, the tissue was washed in 0.1 M phosphate buffer and postfixed in 1% osmium tetroxide for 2 hours. Subsequent to contrasting in uranyl acetate over night, the tissue was dehydrated in ethanol and embedded in Durcopan (Fluka). 1 µ m thick semithin sections were cut using the ultramicrotome and examined by light microscopy (figure 1). The selected and comparable regions of the semithin sections were trimmed and 70nm thick ultrathin sections were made, contrasted with lead citrate and uranyl acetate and examined by electron microscopy on transmission electron microscope Zeiss902A (2) (figure 2). The described procedure enabled the comparison of the brain ultrastructure of mice with different genetic background. The comparison was complemented with the photographing of the serial frames of the selected regions in order to increase the size of area for analysis and acquire the necessary data to be used in image analysis applications. Nevertheless, the variability of the tissue examined should be add to the variability among the mice belonging to the same genotype. The presented example highlights the difficulties of morphology analyisis of the genetically modified mice.

brain; histology; electron microscopy; mouse

nije evidentirano