Updated 7/21/22 by Patricia Graham
Drosophila Fixation Protocol
1) For each sample set up:
a. 1.5ml tube with 500ul 4% paraformaldehyde in PBS(T) + 500ul heptane in the hood.
i. For large samples use 3-5ml heptane and an equal volume of 4% PFA in a scintillation vial.
ii. Dilute the 12% PFA (stored in the freezer) with PBS or PBST to get 4%. Keep that in the refrigerator. It is good for about a month when working with Drosophila. It may be that you need fresh PFA with other insects.
b. Mesh basket in a small beaker near the sink
c. 10ml of bleach near the sink. For Clorox bleach use 4ml bleach plus 6 ml water (10 ml total) per sample.
2) Wash the eggs off the plates and into the basket with cold water.
a. Use a paintbrush to “moosh” the yeast and gently loosen the eggs from the plate.
Try to avoid gouging the agar as agar bits make life complicated later.
b. It will probably take 3-5 rounds of rinsing to get most of the eggs
3) Rinse the eggs 3-5 times with water to remove the yeast.
4) Replace the basket in the empty beaker and add 10ml bleach solution. Incubate 3 minutes, swirling occasionally to remove chorion. Rinse thoroughly with water. Try to get the embryos into the middle of the mesh as much as possible.
5) Remove the mesh carefully from the basket with tweezers. Gently squeeze it in half and submerge it in the solution of heptane:PFA in the 1.5ml tube. Swish it around a bit and pull it out. If there are lots of eggs on the top half, invert the mesh and repeat.
6) Incubate on the shaker for 8-15 minutes (If looking at GFP, use 8 minutes. Otherwise go 15).
7) Allow to settle briefly, then remove as much of the bottom layer as possible without disturbing the interface. Place it in the PFA waste bottle. Most of the eggs should be at the interface, avoid removing them. There may be a little white precipitate at the bottom. This is normal.
8) Add a volume of MeOH equal to the volume of heptane in step 1a, and shake vigorously for 15-20 seconds.
9) Allow to settle (tap gently a time or two to loosen eggs on the sides), then remove the top layer and put it in the 1:1 heptane:MeOH waste bottle.
10) Remove most of the rest of the liquid, leaving the embryos. Put the liquids you remove in the 1:1 heptane:MeOH waste bottle. Most of the eggs should be on the bottom.
a. If using a scintillation vial, transfer the embryos to a 1.5ml tube.
11) Add 800ul MeOH again and invert several times. Allow to settle, remove MeOH and place in 1:1 heptane:MeOH waste.
12) Rinse 2X more with 800ul MeOH (as in step10), but put the MeOH in the 100% MeOH waste.
13) Add 500-800ul MeOH and store at -20oC.
14) Clean up – rinse the mesh, and collection basket and beakers to remove PFA and any embryos that stuck from the mesh. Put them to the side of the sink to dry. Remove the agar from the plate and put it in the trash, then put the empty plate (top and bottom) in the tupperware tub with soapy water in the sink.
NOTE: If you want to stain with phalloiden, you must not use MeOH! Substitute EtOH for MeOH in the above protocol.
Short version
1) Bleach to remove chorion (3 minutes in 50% bleach)
2) Fix in 1:1 heptane:4% PFA in PBS(T) for 8 or 15 minutes
3) Remove bottom layer. Shake with MeOH 15-20 sec.
4) Remove top, then bottom layers.
5) Rinse 3X with MeOH.
6) Store in MeOH.
Drosophila Fixation Protocol
1) For each sample set up:
a. 1.5ml tube with 500ul 4% paraformaldehyde in PBS(T) + 500ul heptane in the hood.
i. For large samples use 3-5ml heptane and an equal volume of 4% PFA in a scintillation vial.
ii. Dilute the 12% PFA (stored in the freezer) with PBS or PBST to get 4%. Keep that in the refrigerator. It is good for about a month when working with Drosophila. It may be that you need fresh PFA with other insects.
b. Mesh basket in a small beaker near the sink
c. 10ml of bleach near the sink. For Clorox bleach use 4ml bleach plus 6 ml water (10 ml total) per sample.
2) Wash the eggs off the plates and into the basket with cold water.
a. Use a paintbrush to “moosh” the yeast and gently loosen the eggs from the plate.
Try to avoid gouging the agar as agar bits make life complicated later.
b. It will probably take 3-5 rounds of rinsing to get most of the eggs
3) Rinse the eggs 3-5 times with water to remove the yeast.
4) Replace the basket in the empty beaker and add 10ml bleach solution. Incubate 3 minutes, swirling occasionally to remove chorion. Rinse thoroughly with water. Try to get the embryos into the middle of the mesh as much as possible.
5) Remove the mesh carefully from the basket with tweezers. Gently squeeze it in half and submerge it in the solution of heptane:PFA in the 1.5ml tube. Swish it around a bit and pull it out. If there are lots of eggs on the top half, invert the mesh and repeat.
6) Incubate on the shaker for 8-15 minutes (If looking at GFP, use 8 minutes. Otherwise go 15).
7) Allow to settle briefly, then remove as much of the bottom layer as possible without disturbing the interface. Place it in the PFA waste bottle. Most of the eggs should be at the interface, avoid removing them. There may be a little white precipitate at the bottom. This is normal.
8) Add a volume of MeOH equal to the volume of heptane in step 1a, and shake vigorously for 15-20 seconds.
9) Allow to settle (tap gently a time or two to loosen eggs on the sides), then remove the top layer and put it in the 1:1 heptane:MeOH waste bottle.
10) Remove most of the rest of the liquid, leaving the embryos. Put the liquids you remove in the 1:1 heptane:MeOH waste bottle. Most of the eggs should be on the bottom.
a. If using a scintillation vial, transfer the embryos to a 1.5ml tube.
11) Add 800ul MeOH again and invert several times. Allow to settle, remove MeOH and place in 1:1 heptane:MeOH waste.
12) Rinse 2X more with 800ul MeOH (as in step10), but put the MeOH in the 100% MeOH waste.
13) Add 500-800ul MeOH and store at -20oC.
14) Clean up – rinse the mesh, and collection basket and beakers to remove PFA and any embryos that stuck from the mesh. Put them to the side of the sink to dry. Remove the agar from the plate and put it in the trash, then put the empty plate (top and bottom) in the tupperware tub with soapy water in the sink.
NOTE: If you want to stain with phalloiden, you must not use MeOH! Substitute EtOH for MeOH in the above protocol.
Short version
1) Bleach to remove chorion (3 minutes in 50% bleach)
2) Fix in 1:1 heptane:4% PFA in PBS(T) for 8 or 15 minutes
3) Remove bottom layer. Shake with MeOH 15-20 sec.
4) Remove top, then bottom layers.
5) Rinse 3X with MeOH.
6) Store in MeOH.