DAPI-staining of sea urchin embryos

(This protocol originated from Eric Cole in the Biology Dept. at St. Olaf College, MN)

1. Spawn eggs and sperm by injecting 0.5M KCl para-orally. Collect eggs by inverting female urchins over artficial sea water in 100ml beakers. Collect sperm "dry" by inverting over small plastic petri dishes.

2. De-jelly eggs by passing them through 68 µm Nitex mesh 2-3x. (Use a rubber band and cut off 60ml syringe to hold mesh.) Confirm viability by adding sperm to a small aliquot. Do you see fertilization envelopes?

3. Prepare dilute sperm solution (1-2 drops in 100ml sea water). Add sperm to fertilize. View eggs after 1 minute. If low rate of fertilization, add 1-2 more drops to sperm solution. Add sperm solution to eggs. Recheck for % fertilization.

4. Meanwhile, prepare polylysine slides. Add ~20µl poly-L-lysine (0.1%) solution to a well on 3-well slide. Spead over well evenly and let dry. Mark the well that is subbed.

5. At appropriate time(s), allow eggs to settle, pipette a drop of concentrated eggs onto a prepared polylysine coated slide to create a monolayer. Let eggs in drop settle for 20-30 seconds.

6. Gently drain excess sea water onto blotter paper (or kimwipe), and immerse slide in Extraction Buffer for 15 minutes (room temp). (Use coplin jars. For this and all subsequent steps, keep cell monolayer FACING you.) This step permeabilizes zygotes and removes some cytoplasmic components that autofluoresce.

7. Drain excess buffer and transfer coverslip to ice-cold 100% methanol. Place in freezer for 15-30 minutes to ensure complete fixation.

8. Dip slides into PBS with 0.1% Triton-X100 with DAPI (5 µg/ml final concentration). Incubate 2-3 minutes.

9. Rinse 3 x in PBS, 5 minutes each.

10. Immerse slides in dH20 for 2-3 seconds to remove excess salts.

11. Place coverslip on well. Seal with nail-polish if desired. Observe using fluorescence microscopy.

 

0.5 M KCl

MW = 74.55 g
18.6 g per 500 ml dH2O

 

Artificial Sea water (MBL- Wood's Hole recipe; Leland Johnson)

24.72 g NaCl
0.67 g KCl
1.36 g CaCl2·2H2O
4.66 g MgCl2·6H2O
6.29 g MgSO4·7H2O
0.18 g NaHCO3

Add dH2O to make 1 L

(Note: Add salts one at a time. Always add sodium bicarbonate last.)

 

Extraction Buffer (To make one liter):

5.98 g Hepes
0.1017g MgCl2·6H2O
3.804 g EGTA
20 mls Triton X-100
250 mls glycerol

Add dH2O to make 1 L

pH = 6.9 (store at room temp)

 

PBS (phosphate buffered saline) (very similar to PBS used in C. elegans protocol)

8.0 g NaCl
0.24 g KH2PO4
1.44 g Na2HPO4
0.2 g KCl

Add water to make 1 L.

Adjust pH to 7.4 with HCl.

Add 1 ml of Triton X-100 per L of DAPI-PBS mixture. Omit Triton for post-staining rinses.