RUVKUN Antibody Staining Protocol revised 10/29/90

Fixation:
1. C. elegans N2 grown in liquid or plates. Harvest worms.
Wash for 2 hours at room temp. in 1X M9 or PBS.

2. Fix in 5 ml of FRESH THAT DAY 1% paraformaldehyde:

to which add 3 ml heptane or H2O (heptane not necessary)
and 1 ml 90% methanol;
0.25 M EGTA pH 8.0

0.05 g paraformaldehyde
80 mM KCl
20 mM NaCl
2 mM EGTA
0.5 mM spermidine HCl
0.2 mM spermine
0.5% BME
15 mM PIPES pH 7.4

 

3. Worms put on ice for 10 minutes in order to stop pumping prior to fixation. Worms plus above emulsion shaken forcefully for 10 minutes at room temp.. Frozen in dry ice/ethanol. Immediately defrosted under warm tap water or stored at -70 C for later use.

4. After warming, emulsion shaken for another 60 minutes at RT.
Specimens pelleted in clinical centrifuge, don't spin hard.

5. Wash worms once (in microfuge tube) with Tris Triton buffer:

REDUCING DISULFIDES TO -SH:
6. Incubate in Tris Triton buffer + 1% BME, 37 C, more than 2 hours with agitation. (Some think BME step isn't necessary.) After this point the worms are fragile and shouldn't be spun hard: approximately 1000 rpm. Wash worms once in 1X BO3 buffer. 100X BO3 buffer ( pH 9.2):

7. Incubate in BO3 buffer + 10 mM DTT, 15 minutes, 37 C with agitation. Wash once in 1X BO3 buffer.

OXIDIZE -SH GROUPS TO -SO3:
8. Incubate in BO3 buffer + 1% H2O2, 1 hour, RT. Agitate gently but keep tubes upright because cap can pop open from O2 pressure. Wash once with BO3 buffer and once for 15 min. or more with Antibody Buffer B:

9. Store worms in Antibody Buffer A:

10. Incubate a small aliquot of worms in 10ug/ml RNAse A, 1 hour, 37 C. Stain RNAsed and unRNAsed aliquots (small amts!, don't use up sample) with 1% toluidine blue. Rinse several times in buffer B. Compare two samples at 50X under dissecting scope. If animals are " open" , + RNAse animals will have very little staining of internal tissues.

ANTIBODY INCUBATIONS:

11. Can preincubate in Buffer A (1% BSA) at RT for a few hours.

12. Spin down worms gently and incubate with 1 in Buffer A (30 ul affinity purified anti-lin-14 antibody -preadsorbed with 292 vector protein- straight or diluted 1:1 or 1:2) for 24 hours at RT.

13. Wash for 4-24 hours at RT in Buffer B (usually do 5 quick washes and then 2-3 hours later do 1-2 more).

14. Incubate with 2 in Buffer A, 4-24 hours, RT (1:200 FITC-coupled goat anti-rabbit antibody for lin-14). (2 should be preincubated at RT for 4-24 hours with fixed C. elegans to remove cross-reacting antibodies.)

15. Wash for 4-24 hours at RT in Buffer B (usually a lot of washes for final step (about 8-10X). Store in Buffer B.

PERMANENT SPRINGTIME MOUNTING:

16. Make a pad on a slide of Permanent Springtime Agarose:

17. Put a 3-5 ul drop on the pad of NPG solution (keeps 1-2 months at 4 C):

18. Cover with a coverslip and examine using epiflourescence. After an hour or two the DAPI stain fades to yellow and " bleeds through" to FITC channel, so don't mount too many samples at once. Store stained worms in aluminum foil covered eppendorfs at 4 C. If need to keep slides, do the same.

PHOTOGRAPHY:

19. Kodak TriX400 black and white film or
COLOR: Kodak TMY 5053 or Fuji 400 color film both pushed 2 f-stops in development.

NOTES:

 

 

To make solution:
1st dissolve dry paraformaldehyde in 2 drops NaOH and heat in 60 -70 C bath. Then add other stuff to final concentrations shown above (total volume= 5ml). Then add heptane(or water) and MeOH; EGTA.
• 100mM Tris Cl pH 7.4
  1% Triton X-100
  1 mM EDTA
  1M H3BO3
  0.5 M NaOH
  0.1% BSA
  1X PBS
  0.5% Triton X-100
  0.05% Na Azide
  1mM EDTA
  1% BSA
  1X PBS
  0.5% Triton X-100
  0.05% Na Azide
  1mM EDTA

TO CHECK THAT WORMS ARE PERMEABLE TO MACROMOLECULES:
• 50 mM Tris Cl pH 9.5
  5 mM MgCl
  2% agarose
  -dissolve 2 mg n-propyl gallate in 70 ul glycerol (65 C)
  -add 30 ul 100 mM Tris Cl pH 9.5
  -add 0.2 ul 1 mg/ml DAPI or Hoechst stain (wear gloves, CARCINOGENS)
  Add an equal volume of stained worms and mix with pipette tip.
  -use nail polish to seal slides if need to keep
  -slides can be kept about 2 days at 4 C without sealant
  -n-propyl gallate is to keep fading down and is best for FITC conjugated 2 s (I think). Another antifader is p-phenylnendiamine which is a carcinogen (see ANTIBODY MANUAL).
  -don't use glass tubes or pipettes since worms stick to them