BrdU Labelling of embryos 


Labelling

  1. collect, dechorionate and wash embryos in a net
  2. air dry embryos within the net for about 3 to 5 minutes depending on the humitity. Wipe away any liquid from the bottom of the net with a piece of paper. Set the basket upside down on the bench top. Check the dryness of the embryos under the stereomicroscope. Embryos have a dull sheen as opposed to being really shiny when they are properly dry (this is not always obvious). Be careful not to overdry, or they will not pop out of the vitelline membrane after fixation.
  3. Permeabilisation: put the net in a weigh boat and add octane (or heptane) to cover the embryos. Swirl the embryos to the middle of the basket, and then bounce the basket to spread the embryos in a monolayer within the net. Incubate for three minutes.
  4. Transfer to Schneider medium with BrdU. This is the trickiest part. Carefully remove the basket from the octane and blot from the bottom, first by setting the basket on a paper towel and then by wiping with a kimwipe. Do this quicklxy. IMMEDIATELY check the embryos under the stereomicroscope. Watch the octane evaprating. When it is almost gone, or just gone (this can take only a few seconds), put the net into a new weigh boat and add Schneider medium + 1 mg/ml BrdU BY POURING on TOP. If you pour outside the basket and let the media run up from underneath then the embryos will float which is not good. If you have done it correctly the embryos will stay stuck on the basket. Incubate for 15 min or for however long you wish to do the pulse labelling. Be careful not to overdry the embryos when you remove them from the octane. If they wrinkle a little bit they are OK, if they shivel up, then tey are too dry and they will not devittelinise.
  5. Fixation: Remove the net from the Schneider medium 1 min before the end of the pulse labelling and place into a scintillation vial with heptane. Squirt the embryos off the bottom of the net with heptane. They should come off fairly easily. If they are cemented on there, then they are too dry. Fix as usual in a scintillation vial with 4% or 8% formaldehyd in PBS/heptan.
  6. remove the fixative, add MeOH and shake to devittelinise. Take only the ones that sink to the bottom of the vial. Alternatively you may manually remove the vitteline membrane.
  7. wash 3x with methanol, store at -20°C


    • Detection


    2.  1.        wash with methanol, add 2 M HCl with 0.1% Tween, incubate for 40 min wash 2x in 0.1 M Na-borate for 2 min
    3. wash 3x in PBT, for 3-5 min
    4. incubate with the BrdU antibody for 1 h at RT or overnight at 4°C. Dilute the antibody as recommended. Use a small volume for the staining, like 100 µl, since a large volume may result in strong background staining.
    5. wash 3x10min with PBT
    6. incubate with the secundary antibody.
    7. wash 3x10 min in PBT, counterstain for 1 min with DAPI or Hoechst, wash and mount.



    BrdU stock:  10 mg/ml in PBS (MW = 307,1 g/mol), store in aliquots at -20°C