Embryo Antibody Staining

Embryo Staining protocol

(according to Sarah, who modified Simona’s, who adapted it from Ian)

 

NOTE: read through protocol before beginning! Pay attention to step 7!!!

 

Day 1:

 

1.    Wash off several plates of gravid (preggers) worms with M9 into 1.5 mL microcentrifuge tubes. If you are feeling lucky, you can just do one plate, but 2-3 is best, especially if looking for extra-chromosomal worms.

 

2.    Wash 3-5 times with 1mL M9, spinning in armadillo (30sec-1min) between each spin and aspirating down to ~100uL (be careful not to suck up your worms!!)

 

3.    Add 1mL of fresh bleach solution to tube. Vortex/shake for 2mins. Spin at 8000 RPM for 1 min. Aspirate off bleach solution and add another 1mL of new bleach solution. Vortex/shake 1min. Spin again at 8000 RPM for 1min. Aspirate off bleach solution and add 1mL of M9 (the worms should not spend any longer than 10 mins in the bleach so work efficiently during this step)

 

BLEACH SOLUTION (make fresh each time)

-         8mL dH2O

-         1mL bleach

-         1mL 10M NaOH

 

4.    You should now have embryos (can verify by looking at tube under the scope… if you don’t see any, there is no point in continuing – it will only bring heartache) 

 

5.    Wash embryos 3-5 times with 1mL M9 (don’t cheap out on this step, you don’t want any residual bleach left) Can do quick spin between each wash on the armadillo, aspirating down to ~100uL. For final spin, use centrifuge: 8000 RPM, 1 min and aspirate down to ~100uL.

 

6.    Add 200uL of 2X Witches Brew to each tube as well as 2% BME (2uL) and mix. Make sure you do this in the fume hood and glove up – this stuff stinks and it very hard to get off your hands.

 

7.    Add 100uL of 10% paraformaldehyde to the tubes, invert a couple of times and then drop tubes in liquid nitrogen for 10mins.

 

10% PARAFORMALDEHYDE (lasts about a week, don’t skimp out and use old stuff – it won’t work as well). In a 15mL conical:

-         10 mL dH2O

-         1g paraformaldehyde

-         50uL 10M NaOH

dissolve in 65° water bath (just crank up the 42° water bath, but remember to turn back down). It takes about 30 minutes to dissolve so make sure you complete this step before starting the protocol!)

 

8.    Put tubes in -80° freezer overnight. (I’m not sure why it has to be O/N, but Simona swears by it)

 

Day 2:

 

1.    Take tubes out of -80° freezer and let thaw on ice 2-3 hours.

 

2.    Wash 2X with Tris-Triton Buffer (1 mL each time). Spin at 8000 RPM, 1min between each wash. Aspirate down to ~100mL.

 

3.    Add 1mL PBST-B and let rotate for 15mins at RT.

 

At this point you can aliquot out 500uL samples of your embryo suspension if you need to do various staining, or you can store at 4° for later use)

 

4.    Spin and aspirate off PBST-B. Wash 1X with 1mL PBST-A and aspirate down to ~30-50uL (be careful not to suck up embryos!!)

 

5.    Add primary antibody (generally a 10 fold increase from what works on western blots: ie. If 1:250 on western, 1:25 for embryo staining).

 

6.    Let sit O/N at RT. (I have let it rotate O/N and it seems to work well)

 

Day 3:

 

1.     Wash embryos 4X with PBST-B, rotating (1mL, 25 mins between each wash)

 

2.     Wash 1X with 1mL PBST-A, spin and aspirate down to 500uL.

 

3.     Add secondary antibody (usually 0.5-1uL/ 500ul).

 

4.     Let incubate 2-3 hours in the DARK!! - wrap in foil. (I have been having more success with letting the embryos incubate O/N, but use your discretion). Again, I have had equal success with just letting them sit as well as rotating.

Ian's edit: you don't need to leave 2o antibody overnight (30 min to an hour is good enough) and you don't need to rotate.

 

Day 3 cont’d or Day 4:

 

1.     Wash 4X with PBST-B, rotating (1mL, 25 mins between each wash). If you incubated with secondary antibody for 2-3 hours, this can be reduced)

 

2.     Spin down and aspirate off as much as possible (30-50uL) to get a high concentration of embryos on the microscope slide

 

3.     Flick tube a couple times to resuspend embryos and put ~9uL of embryo suspension on microscope slide and 11uL of Pro-Long Gold Anti-fade solution (in white plastic cylinder in -20° freezer). If you have a low concentration of embryos, don’t resuspend and just draw directly from the bottom of the tube. Try not to exceed 20uL total on the slide otherwise it takes too long to dry and your embryos slide around inside. Cover with cover slip and let dry 5 mins, seal with clear nail polish. Store slides in the dark.

 

 

GOOD LUCK!!!