This protocol was provided to us from the Grainger lab and modified by M. Khokha to include the lead brick for better squashing.
1. Place 10 Nieuwkoop & Faber Stage 26-34 tadpoles into a dish of deionized, distilled water.
2. Remove the yolky ventral portion of the tadpole and discard. Below is a drawing of a stage 30 tadpole with the "yolky ventral portion" outlined in red.
3. Place all dorsal portions together in the DI water and allow to stand for 20 minutes.
4. Pipette the dorsal halves, carrying as little water with them as possible, into an Eppendorf tube containing 0.2 ml of 60% acetic acid in water.
5. Allow to stand for 5 minutes.
6. Pipette all of the tissue, carrying as little acetic acid with it as possible from the tube and place on a positively charged slide (ex. Superfrost Plus from Fisher).
7. Blot away excess acetic acid if necessary.
9. Lead brick modification
Place a large coverslip on the slide. Fold a paper towel so that it is about the size of the coverslip and place it on top of the coverslip. Then place a lead brick on top of the paper towel/coverslip for about 5 minutes. Heavy compression is important for getting good spreads. However, the coverslip should not slide around. Preventing the coverslip from sliding is crucial in order to get high quality spreads. This is particularly important when placing the coverslip on the slide and when removing the lead brick and paper towel. After five minutes carefully remove the lead brick and paper towel.
10. Place slide and coverslip on dry ice for 5 minutes.
11. Remove slide and coverslip from dry ice and use a razor blade to gently pry up the edge of the coverslip from the still frozen slide.
12. Remove coverslip.
13. Place slide on a paper towel and stain the nuclei/chromosomes by covering the slide with Hoechst 33342 [1 microliter Hoechst 33342 (stock: 0.1mg/ml) in 1 ml distilled water] for 5 minutes. Wear gloves when working with Hoechst!
14. Tip the slide up and allow stain to run off the slide and onto the paper towel.
15. Mount by placing a drop of PBS/glycerol on slide, add large coverslip and seal edges with clear nail polish.
16. Examine slide for presence of stained chromosomes using UV fluorescence using a high power (63X or higher) objective.
17. If done properly, this technique should result in hundreds, possibly thousands, of spread chromosomes. Observing countable spreads requires patience, but most slides yield several countable spreads.