Dissecting and Immunostaining Drosophila melanogaster Egg Chambers
The fix : 4 % Paraformaldehyde , 1X PBS .
Place 10 ml of 1X PBS in the microwave just to boil .
Add the . 4 paraformaldehyde .
Add 4 drops of 2M NaCl ( make sure that Paraphormaldehyde is completely dissolved ) .
Add one drop 12ml HCL to adjust the PH ( 7.5 - 8 ) .
The block : 10 ml of 1X PBS 10µl / ml Normal Donkey Serum 10µl / ml 10X Triton .
Set up the dissection microscopy .
Put the depression slide on it .
Put three drops of 1X PBS on the slide to maintain the PH and keep the egg chambers hydrated .
Anesthetize the flies by using CO2 bed .
Hold the fly from its wings with a size 5 tweezer .
Place it on the slide .
With your left hand hold the fly from its thorax and with your right hand gently grap the tip of the abdomen and pull it out ( be sure to not damage the ovaries ) .
Squeeze the abdomen gently until the pair of the ovaries comes out .
Transfer the ovaries from the depression slide into the fix using transfer piptte .
Wash the ovaries 3X with 1X PBS using transfer piptte .
Transfer the ovaries into the block using the transfer piptte .
After blocking the ovaries , transfer them into a microtube that is filled with fresh block add the primary antibody over night at 4C .
Next morning :
Remove the block .
Wash the ovaries 3X 1X PBS .
Add block Rock at RT for 20 mins .
After 30 mins .
Remove the block .
Wash one time with 1X PBS .
Add block .
Add secondry antibodiy .
Be sure to cover the tube with foil to prevent the interaction between 2nd antibody with the light .
Rock at RT for 2 - 4 hours .
After secondray staining :
Remove the block .
Wash 3X with 1X PBS .
Add block .
Rock for 30 mins at RT After washing the non - specific secondary antibody :
Wash one time with 1X PBS .
By using glass piptte , transfer the ovaries into a regular slide .
Try to remove most of the PBS but leave a little to keep the egg chamber moisturized .
Put three drops of mounting media and place the cover slip on top of the ovaries .
Heal the edges of the cover slip with a nail polish .
The ovaries are ready to be visualized .
