Lessard Group Protocols
Tissue isolation and embedding in OCT (Optimal Cutting Temperature Compound)
- Dissect tissue (e.g. bladder, ureter, etc.) rapidly and place each specimen in OCT using a plastic mold (Fisher Scientific).
- Be sure the specimen is covered in central position, not too near the top and in the desired orientation for sectioning.
- Immediately freeze the sample by placing it on dry ice.
- When the OCT is completely frozen keep the mold in dry ice, and then store long term in liquid nitrogen freezer or at –80oC.
- Place mold in the cryostat chamber for a several minutes to equilibrate temperature. We routinely section at –18oC to –23oC. Place OCT on the chuck and let it cool but not freeze. Remove the block from the mold, place it on the chuck, and let it freeze in position. If the location of the tissue is known, the block can be trimmed with a fresh razor blade to minimize the size of the section. Trim the surface of the block in 50 micron slices until the tissue can be seen. If necessary, trim the surrounding block with a razor blade at this time.
- Cut 9 micron sections for the Arcturus Veritas LCM. Collect specimens at regular intervals on glass slides and inspect them to determine whether the tissue of interest is present.
- Use PEN (Polyethylene Napthalate coated) glass slides (Arcturus) to collect the sections of interest. Space the sections so that the collector cap can rest with one tissue section centered, and with no other tissue section under the edges. For example, we put 5-7 sections of newborn bladder on a slide.
- As soon as the sections are collected, place the slides in a slide box with dry ice.
- Store the slides at –80oC for up to two days until prepared for LCM.
Laser Capture Microdissection (Adapted from the Arcturus protocols)
- Remove slides to be used from –80oC freezer to dry ice. Place on a Kimwipe or paper towel to thaw for 30-60 seconds. From our experience, about 1-4 slides will require about 1 hour of LCM time. More time is required for compartments/structures that are small, less time is required for lower resolution (and/or more highly abundant compartments). A good rule of thumb is that no more than 4 slides be prepared at a time.
- If sampling will be based on EGFP or EYFP expression, the signal can be enhanced by fixing the section for 5 minutes in 4% paraformaldehyde in phosphate buffered saline at room temperature. Rinse for 30 seconds in DEPC treated distilled water.
- Whether paraformaldehyde fixed or not, place the slice in 75% ethanol for 30 seconds.
- Place the slice in DEPC water for 30 seconds.
- (Optional, depending on the criteria for capture) Stain the sections using Histogene (Arcturus). Place the slide on a Kimwipe and cover the sections for 20 seconds with about 200 microliters of Histogene.
- Wash for 30 seconds in DEPC water.
- Dehydrate by incubating for 30 seconds each in 75% ethanol, 95% ethanol and 100% ethanol.
- Place in xylene for 5 minutes. Remove and air dry for 5 minutes.
- Put the slides in a slide tray within an airtight container with a drying agent (DriRite).
- Capture specimens onto LCM caps using the Arcturus Veritas LCM according to their protocols (www.arctur.com). In brief, the compartment of interest is identified based on staining, expression of a fluorescent marker, or other criteria. A UV laser cuts around the specimen and an IR capture laser fuses (spot welds) the cut region of the PEN surface to the cap to capture the specimen away from the slide. The cap is stored on a micrfuge tube in liquid nitrogen until used to isolate the RNA.
Qiagen RNeasy Microkit (c.f. Potter).
EpiCentre TargetAmp 2-round aminoallyl-aRNA Amplification kit. (c.f. Potter)
Affymetrix Gene Chip Mouse Genome 430 2.0 Array (c.f. Potter)