Laser Capture Microdissection to Isolate Purkinje Cells from a Human Post-Mortem Cerebellum Tissue

Take a human postmortem cerebellar tissue containing the Purkinje cell, granule, and molecular cell layers.

Place it on a cryostat sample holder containing partially frozen embedding media.

Allow the media to freeze completely, securing the tissue.

Transfer this assembly into the cryostat cutting arm and allow the tissue to acclimatize to the cryostat temperature.

Move the tissue closer to the blade and trim it to expose the cortex layers.

Place an anti-roll plate above the blade and obtain cerebellar sections.

Transfer these sections to a membrane slide.

Incubate in increasing ethanol concentrations for tissue fixation and dehydration.

Then, incubate in xylene.

Dry the slide and place it on a UV laser capture microscope stage.

 Position the collection cap over the cerebellar tissue.

Microscopically, focus on the Purkinje cells.

Using the laser, capture and collect the Purkinje cells for further analysis.

First, cut a small piece of tissue for sectioning, making sure that the section is approximately 95% cerebellar cortex. Return the remaining tissue either to dry ice or to a freezer at minus 80 degrees Celsius. Next, begin slowly adding OCT on top of the chuck with a slow circular motion. Build up layers of OCT until there is a mound, approximately three millimeters high covering the chuck.

When the OCT is partially frozen, but still has some liquid in the center, place the tissues on top of the mound. Let the tissues sit on top of the OCT for 1 to 2 minutes until it is completely frozen. Then, transfer the chuck with the frozen OCT and tissue into the cryostat cutting arm. Adjust the tissue so that it is flush with the cutting blade, and let the tissues sit in the cutting arm for 20 minutes to adjust to the new temperature.

The most critical step is allowing the tissue to acclimate in the cryostat cutting arm for as long as necessary. If tissue shreds, Purkinje cell visualization will be very difficult. I recommend using smaller, thinner pieces of tissue to allow the tissue to acclimate faster.

After this, slowly move the tissue closer to the blade. Once the tissue has reached the blade, begin the trimming process. Trim the tissue 2 to 3 times until the cortex layers are visible.

Next, place and align the anti-roll plate just above the cryostat blade. Cut 4 to 6 sections that are 14 micrometers thick, noting that properly cut sections will be flat under the anti-roll plate. Align the cut sections horizontally across the cryostat stage.

Angle a slide to pick up all of the tissue pieces, simultaneously. Immediately after sectioning the tissue, place the slide in a slide holder with 70% ethanol on ice for 2 minutes. Transfer the slide to a slide holder with 95% ethanol on ice for 45 seconds.

Next, place the slide in a slide holder with 100% ethanol at room temperature for 2 minutes. Dip the slide 3 times into the slide holder containing xylene number 1 at room temperature. Then, place the slide in a slide holder with xylene 2 at room temperature for 5 minutes. Stand the slides up in a clean fume hood and let them air dry for at least 30 minutes.

First, use RNase decontaminated to clean the microscope stage in the cap collection arm. With gloved hands, place the slide on the microscope and a 500 microliter opaque cap in the collection arm. At a low magnification, align the opaque cap over the cerebellar tissue, ensuring that the cap covers the entire area that is visualized in the eyepiece.

Use a 5 times to 10 times objective to visualize the cerebellar layers. Place the cursor over the section where molecular layer and granule cell layer intersect. Move to a 40 times magnification, and visualize the Purkinje cells. Then, begin capturing them.