Preparation of culture dishes
1: Prepare 0.6% Bacto agar in 121 mM NaCl . Cool to 48C in a waterbath and mix with 50ml thin albumin collected with a syringe from fertilised eggs. Poor 3ml of the mix into in culture plates and allow to gel at room temperature (plates can be kept several days at 4C)
2: Prepare circular rings of Whatman filter paper by cutting out the central disk (25mm) and sterilise the rings..
Isolation of embryos
This part of the procedure is analogous to that used in the widely used EC culture protocol 2. We will shortly describe the main features below.
1: Break the eggs in a clean 10cm petridish, keeping the vitelline membrane around the yolk intact.
2: Orient the yolk such that the embryo sits on top.
3: Clean the surface of the vitelline membrane with sterile tissue.
4: Place the filter paper ring on top of the vitelline membrane centring the embryo in the hole of the filter paper ring.
5: Cut vitelline membrane around the outer edges of the filtering with small scissors and gently lift embryo of the yolk with fine forceps.
6: Put embryos with the epiblast side down on the agar albumin filled culture dish and place under a dissecting microscope (fig 2A).
7: Remove excess yolk with fine tweezers
Mounting of embryos on incubation plate (Fig 2)
Figure 2 Procedure for mounting of the embryo on the culture plate
1: Sterilise the culture plates (fig 1) with 70% ethanol and rinse with sterile distilled water
2: Fill central depression of culture plate with 30ul of heavy Silicon oil
3: take the filter paper mounted embryo from the culture plate (Fig 2A) and place it hypoblast side down over the central depression of the culture plate (Fig 2C).
4: Place rubber O ring in the groove of the culture plate and press down gently with the help of two forceps (Fig 2D). We normally cut the O rings which facilitates their mounting in the groove of the culture plate.
5: Remove the filter ring outside the embryo by tearing the vitelline membrane with fine forces
6: Place the culture plate in a sterile 3cm petridish and fill with 3ml of thin albumin and remove any yolk extruding from undernesth the vitelline membrane into the four escape ports.
7: For observation in a lightsheet microscope we mount the culture plate at the bottom of an observation chamber (microscope coverslip box, 7x3x1.8 centimeter, Fig 2E). To immobilise the culture plate we make an agar mould that will fit the culture plate. The mould is prepared in advance by placing an unused culture plate in the centre of the observation chamber and filling it with 2ml 1% agar in saline. After the agar is solidified the empty culture plate is taken out and replaced with the culture plate that has the embryo mounted (Fig 2F)
8: Fill box with 10ml thin egg white containing optionally Pen/Strep to prevent bacterial contamination.
9: Mount preparation under the Lightsheet microscope and cover the albumin with 2 ml light silicon oil to prevent evaporation.