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.
