Baskets for ICC on Small Tissue Nicole Schmitt 7/2/09
The purpose of these baskets is to make life easier when doing ICC on very small tissue such as mouse utricles or mouse cochlea. They minimize loss of small tissue or damage due to excessive handling.
Start with a 0.6 ml Eppendorf tube. Cut off the top and bottom with a new razor blade as shown. Try to make the cuts as straight as possible so that the bottom of the basket will be flat.
You will then be left with a plastic cylinder with a flat top and bottom.
Next, scratch one of the surfaces (bottom) with sandpaper. This will help the glue adhere.
Cut a small (~ 1 cm) square of nylon mesh with ~ 200 mm holes (www.smallparts.com).
Next, apply a thin layer of aquarium silicone (DAP brand aquarium silicone works well) around the sanded bottom edge. Then use your thumb to press the glue-laden end onto the nylon mesh square. Apply pressure for about 10 seconds. Then, leave it in a safe place to dry in this position. Allow the silicone to cure per the manufacturerÕs instructions (usually 24 hours), and then the excess mesh can be gently trimmed with a manicure scissor or other small scissor.
Baskets are then used for ICC in 48-well plates. They fit particularly well in the Falcon brand plates. Simply put the basket into the designated well and add some PBS to fill the basket about halfway, then add your tissue. To move to the next well containing your next reagent, use a forcep to gently grab the edge of the basket, dab it onto a Kimwipe to remove excess liquid, and place it gently into the next well. About 250 microliters of antibody solution is usually adequate for each well.
To retrieve your tissue following ICC, simply invert the basket over a petri dish or other container, and spray the upended bottom of the basket with a stream of PBS. Usually the tissue will fall easily out, though sometimes a bit more coaxing with PBS is required.
Occasionally, a small piece of tissue becomes lodged up against the glue or side wall and will not be adequately exposed to antibodies. To avoid this problem, gently shake each basket with a forcep during critical steps to make sure that all of the tissue is free-floating and adequately exposed.
Following ICC, rinse the baskets thoroughly with ethanol and allow to dry. They can be reused several times before the mesh starts to peel away; gently check each basket to make sure the mesh is still firmly attached before each use.
In the Rubelab, all of the above supplies can be found in histology, in the drawer with the razor blades/keys. Aquarium silicone is available at Home Depot, and nylon mesh of various sizes is available at www.smallparts.com
If any questions arise, contact Nikki Schmitt: schmittn@u.washington.edu.