Sample Mounting

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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.
 
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.
 
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File: Adding_FocusClear.jpg|Adding FocusClear to chamber
 
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Revision as of 06:59, 16 April 2014

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.

Contents

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. (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

Incubate sample in FocusClear

Form a thin putty cylinder

Place putty on glass slide

Place sample on glass slide between putty

Sandwich sample with glass dish/cover slip

Add FocusClear to inside of chamber

apply silicone elastomer to seal chamber