RESUMO
For bone preparation, several maceration techniques are available; however, gentle defleshing and soft tissue removal from bones without damaging the bone surface remains a challenge. The study aims to develop a novel, inexpensive, rapid, and resource-saving maceration technique that does not lead to bone surface changes and allows the study of signs of violence on the bone surface. Pig ribs and femurs were covered in aluminum foil and treated in the oven for 5 h at different temperatures with and without detergent. Additionally, femurs were placed in hot non-boiling water containing household bleach and detergents for 1 h. Examinations using macro photography, stereo and fluorescence microscopy, and micro-computed tomography showed no surface changes after treatment at 100 °C, such as flakes or bone cracks, on the ribs. The femurs showed surface changes after incubation in water, such as roughening.
RESUMO
For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.
