Immunohistochemistry - Drosophila Embryo
Time collection of embryos to obtain the correct stage desired for analysis .
Collection is completed using apple juice agar plates with a small amount of yeast paste applied to surface .
Our collections are typically conducted at 25 degrees C , unless otherwise specified in published materials and methods .
Wash embryos from apple juice agar plate and into an embryo wash basket using a small paintbrush and embryo wash solution .
Place wash basket into a small tray and pour household bleach on top of the embryos until completely covered .
Let sit for 3 minutes to remove chorion .
Rinse bleach from wash basket and embryos .
Once bleach is removed , transfer embryos to container with 50 : 50 4 % paraformaldehyde and heptane .
Shake on orbital rotator at 150rpm for 20 mins .
Allow solution to heptane and PFA to separate .
Remove bottom phase ( PFA ) and dispose of as required .
Be sure to not disturb or remove the embryos that are trapped in the interface .
Add Methanol in equal volume to the remaining heptane .
Shake vigorously for 20 seconds and allow embryos to settle to the bottom of the container .
Remove heptane from the top layer and most of the methanol .
Add methanol and remove two more times .
Transfer embryos in methanol to disposable culture tubes ( flint glass ) and allow to settle .
Remove methanol without disturbing the embryos at the bottom of the tube .
Rinse embryos with 750ul of blocking solution , once .
Allow embryos to settle to the bottom of tube .
Remove blocking solution and rinse twice with 1X PBS .
Remove 1X PBS and add 750ul of blocking solution .
Seal tube with parafilm and rock gently for 30 minutes .
Remove blocking solution and rinse once with 1X PBS .
Remove 1X PBS and add blocking solution .
Add primary antibodies in optimized concentrations .
( See Optimized Concentrations for Developmental Studies Hybridoma Bank Antibodies for use with Drosophila Embryos ) .
Wrap tube with parafilm and rock overnight at 4 degrees Celsius .
Remove blocking solution with primary antibodies .
Rinse three times with 1X PBS .
Remove 1X PBS and add 750ul of blocking solution .
Wrap tube with parafilm and rock at room temperature for 30 minutes .
Remove blocking solution and rinse once with 1X PBS .
Remove 1X PBS and add new blocking solution .
Add secondary antibodies at optimized concentrations .
Our studies used concentrations of 1 : 1000 .
Wrap tube with parafilm and foil .
Rock gently for 2 - 3 hours at room temperature .
Remove blocking solution with secondary antibodies .
Rinse three times with 1X PBS .
Add 750ul of blocking solution .
Wrap tube with parafilm and foil .
Rock for 30 minutes .
Remove blocking solution and rinse once with 1X PBS .
Carefully remove embryos with a glass pipette that has been rinsed with blocking solution and transfer to a microscope slide .
Using Whatman's paper , carefully wick away excess 1X PBS .
Add 35ul of mounting media and cover with coverslip .
Seal with nail polish if desired .
