Agar Embedding

- Agar embedding allows for confirmation of orientation when preparing a specimen for cryostat embedding
- Larval and juvenile zebrafish fixed with Carnoy’s fixative
  • Generally found in cardboard boxes in the freezer
  • Stored with 95% Ethanol
  • Good for antibody staining
  • Specimens fixed in Carnoy’s are usually used in this procedure
- Larval and juvenile zebrafish fixed in 4% paraformaldehyde (PFA)
  • Generally found in plastic boxes in the freezer
  • Good treatment to make proteins stable
  • Specimens fixed in PFA stay in tact during clearing and staining, paraffin embedding and sectioning, and plastic embedding and sectioning (among other protocols)
  • Difficult to use with antibody staining

  1. Using dissecting microscope, sever tails of specimen [only relevant for projects involving head morphology]. Allows for better penetration of solutions
    1. Using forceps, remove fish from its tube container, and place in a petri dish
    2. Gently holding tail end of fish with forceps, use scissors to cut the fish posterior to (behind) the pectoral fin – separating the tail from the head
    3. Delicately grasp the fish head close to where it was cut (watching to not damage the skull) and place back in the original tube
    4. Rehydrate into PBS or PBT
      1. Do this in a series of washes, from 75% to 50% to 25% to pure PBS or PBT
i. In each step, remove current fluid, and replace with appropriate wash
ii. After inserting new fluid, place tube in roller for 15-20 minutes
  1. Size will determine how long this process should take between steps (the bigger the specimen, the more time required)
    1. To double check, look at where the fish is currently located within the tube. If it is floating, it is a safe estimate that it has not yet been saturated with the wash. If it has already sunk to the bottom of the tube, you may be ready to move onto the next wash
    2. Obtain a square plastic mold that will hold ~3mL agar
    3. Prepare 30% sucrose and mark scintillation vial with the original size of specimen (i.e., the label on the specimen’s storage tube), initials and date
      1. To calculate the necessary amount of sucrose, multiply 30% by the amount of distilled water you will be using. Each scintillation vial holds ~20mL. Here, an example if we were to prepare 4 specimens (4 agar blocks), we would need 80mL of sucrose solution.
i. Therefore:
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  1. Using a weigh boat and balance, obtain 24g of sucrose, and place in a graduated cylinder. Fill the rest of the cylinder with distilled water, until you have 80mL
  2. Seal cylinder with parafilm and invert until sucrose has fully dissolved
  3. Pour solution into each scintillation tube, ~75% full (make sure that the addition of agar will not cause a spill)
  4. Melt agar by placing container in a larger beaker containing water. You do not want to boil either substance, only to warm the agar sufficiently so that it becomes liquid (approximately 1 minute at power level 3). Also, it is important that the agar is not too hot so as not to burn the tissue of the specimen.
  5. Obtain specimen and roll on kimwipe to remove excess PBS, and place it into mold
  6. Pipette agar into mold by pouring it into sides to prevent air bubbles, make sure no liquid gets into pipette top (which ruins the pipettor)
    1. Slowly pipetting the agar will also help ensure that there are fewer (if any) air bubbles, and that the specimen will not move around too much in the dish
    2. When the agar begins to set, orient specimen to desired position, then let it harden (about 15 minutes)
    3. Once the agar has fully solidified, lightly bend the edges of mold to release it
      1. Do this gently so that it does not crack, and alternate pressure points
      2. Once agar has separated from the mold, place the mold upside-down on top of a microscope slide
      3. Using a razor blade, cut a trapezoid shape into agar, with the smallest point being at the smallest section of the fish
        1. Example:
Original After Cutting

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  1. Place section in sucrose, store at 4C overnight
    1. Do not start sectioning until the sample has sunken to the bottom of sucrose vial
    2. To ensure infiltration, leaving sample at room temperature overnight is possible, though that may introduce contamination issues later (possible, but not ideal)