No abstract

Ex vivo magnetic resonance imaging (MRI) requires chemical fixation to preserve tissue during storage or extended imaging sessions. Although it is commonly understood that fixation may alter tissue volume and shape, the potential confounding effects of fixation and storage on morphometric analyses have not been well characterized. With increasing use of ex vivo MRI for mouse brain phenotying and opportunities for inter-study comparisons, we sought to characterize how changes in fixation and/or storage times affected tissue volume, and how this might impact phenotyping results. Mouse brain samples that had been perfusion fixed, within the skull as per our standard protocol, were immersed in formaldehyde-based fixative for 1 to 5days before being stored in saline or water. Throughout fixation and storage, samples were repeatedly scanned using magnetic resonance imaging, and analyzed for volume expansion or shrinkage. We found that most of the brain continued to shrink post fixation, with the rate of shrinkage dependent on the solution in which the samples were submerged. Maximum changes in volume of 3.5% per day and 3% per month were detected during fixation and storage (in PBS), respectively. Most notably, changes were non-uniform, with some structures shrinking slower, or even expanding, when compared to other structures in the brain. Our results highlight that caution is necessary when interpreting results from experiments with inconsistent fixation and storage protocols, so as not to mistake these changes for phenotypic differences.

No abstract

It is important to understand changes in cell distribution that occur as a part of disease progression. This is typically achieved using standard sectioning and immunostaining, however, many structures and cell distribution patterns are not readily appreciated in two-dimensions, including the distribution of neural stem and progenitor cells in the mouse forebrain. Three-dimensional immunostaining in the mouse brain has been hampered by poor penetration. For this reason, we have developed a method that allows for entire hemispheres of the mouse brain to be stained using commercially available antibodies. Brains stained for glial fibrillary acidic protein, doublecortin and nestin were imaged in three-dimensions using optical projection tomography and serial two-photon tomography. This staining method is simple, using a combination of heat, time and specimen preparation procedures readily available, so that it can be easily implemented without the need for specialized equipment, making it accessible to most laboratories.

No abstract