REED LAB – Butterfly wing antibody stain protocol
(Adapted from Brunetti et al. 2001. Current Biology, 11:1578-1585)
Fixing Imaginal Wing Discs:
1. Dilute fix in re-usable 15ml conical tube. For each caterpillar add 950ul
of 1.05X PEMT and 50ul 37% formaldehyde (white plastic bottle in
hood). Invert conical a few times to mix.
(eg. If you are dissecting 4 butterflies mix 4x 950ul squirts of PEMT and 200ul of
formaldehyde. Dissect only the number of caterpillars you can complete in 30min)
2. Aliquot 1ml of diluted fix into wells of a 24 well culture plate. Write the
species code for each well.
(eg. “VC” = Vanessa cardui, “JC”=Junonia coenia, “HE”=Heliconius erato)
3. In cold PBS dissect all 4 discs out of a caterpillar as quickly as possible
and transfer them into one well of the plate using a 1000ml pipette tip cut
to enlarge the opening. Minimize the amount of carry-over PBS. Start
timer.
4. Continue dissecting caterpillars until 30min are up. Keep track of how
long each set of wings has been in the fix.
5. After 30min are up for each respective set of discs wash them 4x5min in
~1mL (one pasteur pipette squeeze) ice cold Ab Wash. Put dish on
nutator during washes.
6. After final wash add cold Ab Block.
7. When all discs are in Ab Block, write today’s date on each well and put
plate in refrigerator (4˚C).
(Wing discs may be stored in block for several weeks before an antibody stain. If you
will be storing discs for more than a few days, transfer the discs+block from the
culture plate to microcentrifuge tubes)
Ab Stain Day 1
1. Dilute antibody in cold Primary Buffer in a .6mL microcentrifuge tube.
Set aside for a moment.
(Typical dilutions: Notch = 1:200, cut = 1:200, Dll = 1:100, spalt 1:200, Engrailed
4F11 = 1:5).
2. Place discs in Ab Block in a 1.5mL microcentrifuge tube.
3. Remove Ab Block from the discs with a pipette. Add enough antibody
dilution so the level is a few mm above the wing discs. Add a minimum of
10uL of the Ab dilution.
4. Place tubes upright in a rack at 4˚C overnight.
Ab Stain Day 2
1. Remove antibody from wing discs.
(If the antibody is in short supply, like Distal-less or spalt, keep the used antibody in a
dated tube at 4˚C)
2. Wash discs 4x5min in ~1ml cold AB Wash on nutator.
3. Dilute secondary antibody 1:200 in Ab Wash and apply to wing discs
after removing final wash. Make sure discs are completely submerged.
4. Keep tubes upright in a foil envelope in a drawer for 2-3hr at room
temperature.
5. Wash discs 4x5min in ~1ml cold Ab Wash on nutator. Keep tubes in a foil
envelope the entire time.
OPTIONAL: Add 1ul of DAPI or Hoechst nuclear counterstain to final wash.
6. After final wash add glycerol + n-propylgalate to discs and place at 4˚C.
Check back 1-2hr later to push floating discs back down into the glycerol.
7. Mount discs on slide when you are ready to look at them. Cy2 may start to
fade in a few days, while Cy3 can sit for 1-2 weeks before being mounted.
Recipes for Butterfly Antibody Solutions
250ml of 1.05x PEMT
9.09g PIPES
0.10g EGTA
0.13g MgSO4
26.25ml 10% Triton
add ddH20 to 250ml
pH to 6.9
500ml Ab Wash
25ml 1M Tris
15ml 5M NaCl
2.5ml NP40
add ddH20 to 500ml
pH to 6.8
50ml Ab Block
250mg BSA
add Ab Wash Buffer to 50ml
15ml Primary Buffer
15mg BSA
add Ab Wash to 15ml
Fix
Dilute 37% formaldehyde in 1.05x PEMT at 1:20.
(This will give a final formaldehyde concentration of 1.8%).