Cryo-Planing Biological Specimens
micro-renal morphology. A low- magnification survey image shows a cross section through a glomeru- lus (Figure 3A). A higher-magni- fication image of the same region (Figure 3B) clearly shows details of cross-sectioned mitochondria and villi. An additional kidney speci- men provides a view of a deeply tex- tured villus structure (Figure 3C) and a region of the glomerulus that is dense with mitochondria show- ing several cross-sectioned cristae (Figure 3D).
Discussion Te methodology presented
here enables routine cryo-planing specimen preparation without
the
Figure 2: Cryo-planed Christmas cactus (genus Schlumbergera) leaf. (A) Faced plane that shows mostly intact cells. (B) Intact chloroplast in leaf section. (C) Faced plane that is mostly through cells. (D) Boxed region shown in C including cross-sectioned chloroplast.
need for specialized custom devices. Te mPrep/s capsules require only trivial modifications and, with the Workstation, enable facile orienta- tion of the specimens. Te capsules provided an easy-to-manipulate handle for the entire preparative process. Te capsule “holder” also reduced the potential for specimen damage since the specimen itself did not need to be directly held. Because a standard cryo-microtome was used for cryo-facing, it was easy to view and control the location of the cryo-section plane or face. Finally, because multiple specimens held in mPrep screens may be mounted on a single SEM stub, it was easy to view and record comparison images in the SEM. In the present study, aldehyde-
Figure 3: Cryo-planed canine kidney. (A) Cross section of glomerulus. (B) Boxed region of (A) at higher magnifica- tion. (C, D) Two additional regions shown at higher magnification.
2019 March •
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fixed kidney was used. Fixed tis- sues provide both advantages and disadvantages. It is well known that rapid-freezing of fresh tissue can provide the highest possible morphological preservation, but it is imperative that the tissue be extremely fresh, and logistically this can be difficult to accomplish. Tus, pre-fixed tissue can be advan- tageous. An additional advantage of aldehyde pre-fixation is that this makes the tissue a bit tougher and firmer, which can facilitate dissec- tion and mounting in the desired orientation. In the present study, the kidney tissue was also post- fixed in OsO4
. Tis improves mem- brane fixation and also makes the 23
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