Neuroscientists must section whole brain blocks for post-experiment histology in order to diagnose the location of manipulations in each brain. Neurotrauma or some brain surgeries may damage the structural integrity of slices. At several coronal levels, not all parts of a brain are connected to the center block. Accurate determination of location requires all landmarks, with all parts in their correct location.
- Specimen collection Issues
- How to use Tape Transfer
- The magic
Specimen collection Issues
Frozen tissue specimens intended to be sectioned in a flat plain don’t always cooperate. While the microtomist is expected to preserve the appearance and spatial arrangement that he can see in the uncut block, a given section may have gaps. For example, between cerebrum and brainstem, or bone ends at the joints, there may not be any material connecting the parts in a given thin section. Further, tissue may roll up in scrolls unless an antiroll plate or brush is skillfully utilized, and some tissue, especially poor quality tissue, may crumble, fracture or compress before the blade. Efforts to lift a section may sometimes cause it to fall off the blade and get lost. Tissue arrays with many pieces may not stay together in the original order. Tissue may become folded, wrinkled, or have bubbles under it in mounting to the slide. Or multiple specimens may be sectioned with each pass of the blade, and need to stay in order.
How to use tape Transfer
An easy to use and very reliable solution is available to avoid any of these issues: tape transfer technology. Paxinos (Watson, C., Paxinos G. and Kayalioglu, G, 2009) has used tape transfer in his recent atlas work to ensure that every section is captured, and remains in perfect arrangement.
After a frozen tissue block is faced and oriented in a cryostat, a precut tape window is placed on the frozen face and rolled into contact. A section is then cut below that level; the freshly cut section is still adhered to the tape, still frozen, exactly as it was on the block face. No parts can fall away. No brush or anti-roll plate is required to cut or transfer the section. When sectioning tissue arrays, damaged tissue, or at a level with separated parts, or if it is necessary to capture every section (as in atlas construction), this is especially valuable. It also increases speed and efficiency.
The tape window piece with the section is placed face down on a cold slide inside the cryostat, oriented as desired on the slide, and rolled flat into contact to adhere.
It is then placed under a safety plate, where it is irradiated briefly with long wave UV light to polymerize the adhesive layer on the slide into a hard, solvent-resistant, optically clear plastic, tightly anchoring the section to the slide. Note that multiple tape sections can be placed on one slide before it is irradiated.
The tape window can then be gently peeled away at a reverse direction, and discarded. The section, exactly as it was on the block face, is left adhered to the slide, still frozen. It may then be fixed or stained, and will stay adhered to the slide through any common staining protocol.
The technology magic is that the adhesive that holds the frozen section on the tape is released by this wavelength of UV light, while the optically clear adhesive pre-coated on the slide is activated by that wavelength. This two adhesive system, operating at cryostat temperatures, with opposite response to UV light, guarantees the section goes onto the slide in exactly the shape it was in the tissue block. No rolls or folds, no parts moving out of place, or separated components going to a new orientation. And still frozen, never thawed. The tape can then be gently peeled back away, leaving the tissue in place on the slide. The completed cold slide can then be moved into whatever fixation, staining or fluorescent protocol is to be used.
Note that this technology can be employed on unfixed tissue, leaving antigens in their most active form, for excellent immunostaining, HRP fully active, or on fixed tissue. Unfixed tissue need be exposed only to chemicals necessary for the staining, and is already stretched on a slide, cannot shrink in the horizontal plane. Or the unfixed tissue can be fixed as the next step after mounting.
The cured adhesive on the slide is polymerized and inert, and will not interfere with common staining processes, nor be released from the slide by any chemicals used in common staining protocols.
An innovative method to save much cryotomy time is to use Brain Blockers to hold several frozen brains in the cryostat at once. Each pass of the knife will collect the same level of section from each brain, all positioned perpendicular to skull flat orientation. And the order on the original layout will remain fully preserved if used with the tape transfer system. The Cryojane Tape Transfer System can be installed on most Cryostat models.
Watson, C., Paxinos G. and Kayalioglu, G., The Spinal Cord: A Christopher and Dana Reeve Foundation Text and Atlas, Academic Press, San Diego, 2009.