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Staining of C. elegans using Bouin's fixative

  1. Stage animals to the appropriate stage (keep in mind that you have 30 minutes washing).
  2. Wash animals off in M9 and wash again to get rid of bacteria.
  3. Leave the animals in M9 for 30 min. to pump the bacteria out of their intestines.
  4. Spin down the worms and leave them in 50ml M9. Add 400ml Bouin’s Fixative, 400ml Methanol and 10ml BME.
  5. Rotate tubes at room temp for 30 min.
  6. Freeze in liquid N2. Can store at -80°C here.
  7. Thaw under runnig warm water until the solution just melts. Rotate at room temp for 10 min.
  8. Repeat steps 6 and 7 twice (total time rotating after 1st freeze = 30 min).
  9. Quick wash 3X in BTB.
  10. Wash for 1 hr. in BTB.
  11. Spin, remove supernatant and wash for 1 hr. more in BTB.
  12. Quick wash 2X in BT.
  13. Wash 3X in ABA or PBST (PBS + 0.5% Triton X-100) or PBSTA.
  14. Rotate 2 hrs at room temperature in PBSTA + 10% Serum of the animal the secondary is made in). Can put at 4 degrees O/N here.
  15. Dilute primary antibody to twice the end concentration in 30ml PBSTA + 10% serum. Spin the worms, leave them in 30ml and add the primary antibody solution. Rotate O/N at room temp.
  16. Quick wash 2X with PBST or PBSTA. Wash 4X 10 min. with PBST or PBSTA. In the last wash add 10% serum.
  17. Spin the worms and leave them in 30ml. Add 15ml of each precleared secondary antibody solution. If doing a single labeling, replace one secondary with PBSTA + 10% serum. To preclear secondaries, take a mixed N2 population and follow this protocol to step 14. Then dilute secondary antibodies to eight times the end concentration in 30ml PBSTA + 10% serum. Spin the N2 worms, leave them in 30ml and add the secondary antibody solution. Rotate O/N at room temp covered with aluminum foil.
  18. Rotate the worms for 2 hrs. at room temp with the secondaries.
  19. Quick wash 2X with PBST. Wash 2X 10 min with PBST. Keep tube in darkness.
  20. For DAPI, add PBST + 1mg/ml DAPI. Rotate for 30 min. Spin down and put worms on a slide.
  21. For PI, add PBST + 200mg/ml RNAse A. Rotate for 30 min. Quick wash with PBST. Add PBST + 1mg/ml PI. Rotate for 30 min. Spin down and put worms on a slide.
  22. Coverslip slides with DABCO mounting media (90% glycerol, 2.3% DBCO, 10% PBS).

 

Bouin’s Fixative
15 ml picric acid
5 ml formalin (37% formaldehyde, 12.5% methanol)
1 ml glacial acetic acid

BTB
1X Borate buffer
0.5% Triton X-100
2% BME

50X Borate buffer
1 M H3BO3
0.5 M NaOH
pH to ≥9.6

ABA
1X PBS
1% BSA
0.5% Triton X-100
10 mM NaAzide

PBSTA
1X PBS
1% BSA
0.5% Triton X-100
5 mM NaAzide
1 mM EDTA