From CLARITY Wiki
Before mounting the sample for imaging, incubate the sample in the mounting solution for several hours or overnight (until the sample is visually transparent). FocusClear should be used as the mounting solution for large or whole tissue samples. Suggested methods for mounting large tissue samples (whole brain) and smaller sections (<1 mm thick brain slice) using the imaging chamber supplies are detailed below.
Whole brain mounting for confocal imaging
Form a putty cylinder
Roll a piece of BluTack putty into a cylinder with uniform thickness. The cylinder should be several centimeters in length with a thickness greater than that of the brain sample.
Place putty on a glass dish
Shape the putty cylinder into a horseshoe U shape. Place the putty onto the flat side (outside) of a Willco glass dish. Using a pipette tip, press the outside edge of the putty U down onto the glass dish to help seal it.
Place sample onto glass dish
Using a spatula, carefully place the tissue sample on the center of the dish surrounded by, but not touching, the putty.
- Note: A mouse brain before clearing is shown for the purpose of demonstration.
Sandwich sample with second glass dish
Carefully place the flat glass side of a second Willco dish on top of the putty. Slowly press down evenly on the glass dish, keeping fingers over the putty perimeter, until the glass contacts the top of the tissue sample. Check that both glass dishes are parallel to each other.
Add FocusClear to inside of chamber
Using a pipette, add about 1-2 mL of FocusClear into the opening of the sealed chamber (apply the solution used during incubation). Fill up to the black edge of the glass dish, taking care to not introduce any bubbles into the chamber.
Apply silicone elastomer to seal chamber
Apply the Kwik-Sil PDMS sealant across the opening in the putty, making sure to cover the entire space between the glass dishes for a complete seal. Place the dishes on their side with the putty opening on top and let sit for 20 minutes to allow PDMS sealant to fully harden before imaging.
Thin slice mounting for confocal imaging
A method similar to that used for thick/whole tissue mounting can also be applied to thin tissue slices. A slightly modified version is presented below. Note that the Willco dishes can easily be replaced by glass slides or cover slips.
Form a thin putty cylinder
Roll a piece of BluTack putty into a thin cylinder with uniform thickness. The cylinder should have a thickness about two times that of the brain sample slice.
Place putty on glass slide
Place the putty tube perpendicularly across the glass slide. Using a razor blade, cut off the extra putty from outside the glass slide. Place one tube of excess putty parallel to the first tube on the glass slide. Again, cut off the extra putty from the second tube.
Place sample on glass slide between putty
Carefully pipette a small amount of mounting solution (about 10 μL) onto the glass slide between the putty tubes, making sure it does not contact the putty. Using a spatula and pipette tip, carefully place the sample on top of the mounting solution droplet. Make sure no mounting solution gets on the putty cylinders or the chamber will be leaky.
- Note: A brain slice before clearing is shown for the purpose of demonstration.
Sandwich sample with glass dish/cover slip
Place the flat glass outside of the Willco dish on top of the putty tubes. Slowly press down evenly on the glass dish, keeping fingers over the putty, until the glass contacts the top of the brain sample. Check that there are no bubbles between the sample and the glass slides.
Add mounting solution to inside of chamber
Slowly pipette mounting solution into the small opening on the side of the glass slide (apply the solution used during incubation). Fill the entire space between the glass slide and Willco dish, and avoid introducing any bubbles.
Apply silicone elastomer to seal chamber
Apply the Kwik-Sil PDMS sealant across both openings on the side of the glass slide to create a sealed chamber. Let sit for 20 minutes to allow PDMS sealant to fully harden before imaging.