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Antibody Staining

Antibody staining is commonly used in laboratories to locate specific cellular components or molecules on a specimen.  It usually occurs in a two-step method, where the primary antibody is used to target the wanted molecule, and the secondary antibody has a fluorescent tag to locate the primary antibody.  This process occurs over a period of three days.  Embryos are typically inside of Eppendorf tubes (1.5 mL) for all of the following steps.

Day 1

For fish stored in methanol: Re-hydrate the embryos at room temperature with three successive, 5-minute washes, composed of:

1.) 75% methanol/25% PBS 
2.) 50% methanol/50% PBS
3.) 25% methanol/75% PBS

Then, rinse with 1X PBS + 0.5% Triton-X100 (250 µL Triton-X100 in 50 mL PBS) for 5 minutes at room temperature.  Repeat this step three times while rocking the samples during each wash.  

For fish older than 2 days: Incubate in 0.1% collagenase/PBS (25 microliters/10 embryos) for 90 minutes at room temperature.  Do not rock the tubes. 

Quickly wash twice with 1X PBS + 0.05% Triton-X.

Incubate in PBS/BSA/DMSO/Triton-X100 (PBDT:  bring 1 g BSA to 100 mL of PBS, add 1 mL DMSO, and 100 µL Triton-X100) for 1 hr at room temperature.  Then rock the tubes. 

Incubate in the correct dilution of primary antibody in PBDT at 4.0 C overnight, keeping the tubes rocking. 

Day 2

Wash with 1X PBS + 0.05% Triton-X100 for 1 hour at 4.0 C.  Wash a total of 4 times, 1 hour each.

Incubate embryos in 1 mL of diluted secondary antibody in PBDT at 4.0 C, rocking overnight.  Cover with aluminum foil (assuming you are using a fluorescent secondary that is subject to photobleaching-if not, you can ignore aluminum foil wraps throughout).

Day 3

Wash with PBS for 1 hr at 4.0 C, rocking, 3 times. Keep covered with aluminum foil.

Store in 50% glycerol at 4. 0 C, covered with aluminum foil.