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Clearing & Staining for Larvae
Clearing & Staining Nerve Stain
Clearing & Staining with Muscles
Generalized Clearing & Staining Protocol
Lee Methylene Blue
Whole Mount Antibody Staining
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- 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
Using dissecting microscope, sever tails of specimen [only relevant for projects involving head morphology].
Allows for better penetration of solutions
Using forceps, remove fish from its tube container, and place in a petri dish
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
Delicately grasp the fish head close to where it was cut (watching to not damage the skull) and place back in the original tube
Rehydrate into PBS or PBT
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
Size will determine how long this process should take between steps (the bigger the specimen, the more time required)
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
Obtain a square plastic mold that will hold ~3mL agar
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
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.
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
Seal cylinder with parafilm and invert until sucrose has fully dissolved
Pour solution into each scintillation tube, ~75% full (make sure that the addition of agar will not cause a spill)
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.
Obtain specimen and roll on kimwipe to remove excess PBS, and place it into mold
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)
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
When the agar begins to set, orient specimen to desired position, then let it harden (about 15 minutes)
Once the agar has fully solidified, lightly bend the edges of mold to release it
Do this gently so that it does not crack, and alternate pressure points
Once agar has separated from the mold, place the mold upside-down on top of a microscope slide
Using a razor blade, cut a trapezoid shape into agar, with the smallest point being at the smallest section of the fish
Original After Cutting
Place section in sucrose, store at 4
Do not start sectioning until the sample has sunken to the bottom of sucrose vial
To ensure infiltration, leaving sample at room temperature overnight is possible, though that may introduce contamination issues later (possible, but not ideal)
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