Coverslipping and Sealing Samples for Imaging and Storage

Use this protocol if you need to prepare a specimen for imaging with a microscope that has a small working distance (such as the confocal) and preserve it for storage afterward.
1) Gather the supplies you'll need: (also listed below this image)

• Microscope slides - preferably from a box of plain, precleaned, slides with the dimensions 25x75x1mm
• Plastic pipette (or glass, if you prefer)
• Sharpie or another permanent marker
• Nail polish
• Forceps (with black tape on the handle, which designates use with fixed specimens only)
• Reinforcement labels
• Slide covers (coverslips) - preferably from a box that lists the dimensions as "22x22-1.5um"
• Fluoro-gel

2) Obtain your prepared (fixed, sliced) sample.

3) On a slide, label your initials, experiment, and date with your permanent marker:

4) Place one or two reinforcement labels on the slide. If you choose only one, place it in the center of the slide; if two, place them evenly spaced out. The latter is usually preferred because it saves both slides and storage space:

5) Carefully pipette the sample into the center of the reinforcement label. Pipette off any excess solution surrounding the sample.

6) Slowly squeeze one drop of Fluoro-gel on the inside of the ring so it immerses the sample. Be sure to make the drop as small as possible; too much gel will cause the coverslip and/or sample to slide around. If necessary, pipette off any excess gel and reposition the specimen into the appropriate orientation using your forceps. The image below shows the amount of fluo gel you want to cover your specimen:

Coverslipping:
7) Obtain one coverslip for each reinforcement ring. Be sure you pick up only ONE coverslip, as multiples stacked over the sample may damage the microscope objective when the stage is close to it during imaging.
8) Slowly and gently place one edge of the square coverslip on the slide. The goal is to prevent as many bubbles as possible from collecting under the cover/around the specimen. To do this, lean the coverslip at approximately a 45-degree angle from the slide and quickly let go:

 

Both coverslipped:

9) Repeat these steps for each sample before you image. If you do not plan to image immediately after coverslipping your samples, seal the slides with nailpolish (see Step 10). If you are going to image immediately after coverslipping, do not seal the sample with nail polish until you've finished. The goal is to avoid having wet nail polish near the microscope.
Sealing your slides:
10) The purpose of the nail polish is to seal and preserve your sample for future reference. Therefore, you want to apply the nail polish only around the edges of the coverslip and the adjacent slide. To do so, use the brush to apply the paint on a even layer around the edges so that both the coverslip and the slide are painted:

11) Allow the slide to fully dry. If your sample is light sensitive, keep the sample in a dark place while it's drying (a large petri dish wrapped in tin foil is one good option). Once the slide is fully dry, place it in your slide box for storage in a cold, dark location, such as a freezer for storage of fixed specimens.
NOTE: This protocol applies to tissue sections obtained from cryostatting as well; just start at Step 8, as reinforcement rings are not necessary. You may also want to use a larger coverslip to ensure all the sections on your slide are covered and sealed.Make sure the nail polish is fully dry before using on the microscope. The nail polish can seem dry to the touch after a couple of minutes, but it will get on the objectives if you try to image the slide too soon.