Making Agarose Pad Slides for Through-Focus Microscopy
- Use 70% ethanol to clean both sides of new slides (type with frosted label space) and coverslips (use #18 round coverslips for this work).
Use gloves for the next steps. We use standard lab tape that is ¾ inch wide for step 3.
- For the cover or upper “sandwich” slide, use a cotton swab to apply Rain-X to the side of the slide opposite of the frosted label side. Mark the frosted slide label “Rain-X on opposite side”. After the Rain-X dries, polish the surface with a soft cloth or paper towel (or Kimwipe).
- Apply a solution of sticky silane to the bottom slide. Use the surface that has the frosted label. Use a cotton swab to apply the compound. Let the first application dry and then apply again. After the silane dries, apply one piece of lab tape on each end of the slide and to the same side that has the sticky silane. Leave enough space at the ends of the slide so that the center open space is approximately one inch. Allow silane to dry for at least 10 minutes before pouring agarose to make slides.
Sticky silane recipe: 1ml 100% ethanol, 50 ul 10% acetic acid (from Glacial), 7.5 ul of the sticky silane compound (3-(Methacryloyloxy)propyl]trimethoxysilane)
- Make a solution of 0.8% Agarose in water. Distilled water is best unless the nematodes cannot tolerate it (then substitute tap water). Use 0.1g of agarose and 12.5 ml of water. Only reheat this agarose once (i.e., only one re-use).
- Carefully heat the agarose suspension to boiling (by microwave). Dissolve all pieces of agarose, and then cool in a water bath or bead bath set to about 50C.
- Use a P-1000 pipet (blue tip) set to about 300 ul to dispense the hot agarose solution. Do NOT dispense the entire amount onto the bind silane slide (leaving some in the pipet tip reduces bubble formation). Use a new tip for each slide. Now you must work quickly.
- Hold the Rain-X side of the cover slide on an angle relative to the bottom slide. Touch the Rain-X side of the slide to the molten agarose. Then apply the top slide to the bottom slide, pressing to seat the top slide on the tape of the bottom slide. Some agar will ooze out.
- Let the slide cool. If not using the slide immediately, place it in a sealed container that has wet paper towels on the bottom. They have a limited life span!
- Before use carefully scrape off excess agarose from the edge of the slide with a single-edge razor blade. Carefully (and slowly) slide the top slide off in the direction of the tape.
- Add about 10-15 ul of water to the surface of the slide. Then carefully add nematodes without touching the agarose surface. Once nematodes are in place, add a coverslip that has been treated with Rain-X (lowering the coverslip at an angle). The slide should be ready to use.
- After photodocumentation of the nematodes, use a pick to move each in turn to a small drop of 1X PrepGem buffer. Nematodes can be cut in half (tuberculin needle) and moved to a 0.5 ml tube containing 20 ul of 1x PrepGem Gold DNA digestion buffer for DNA preparation.