Phalloidin Staining and Immunohistochemistry of Zebrafish Embryos

Materials and Reagents

1. Alexa Fluor 488 or 546 Phalloidin (Life Technologies)
   
2. Desired primary antibodies (see Table 1 for information for antibodies commonly used in the Henry Lab)
   
3. Alexa Fluor 488, 546, or 633 secondary antibodies (e.g. goat anti-mouse or goat anti-rabbit secondary antibodies, Life Technologies)
   
4. Vacuum grease (e.g. Dow Corning High vacuum grease)
5. 10x PBS (see Recipes)
   
6. PBS 0.1% Tween-20^® (see Recipes)
   
7. PBS 2% Tween-20^® (see Recipes)
   
8. 8% PFA (see Recipes)
   
9. 4% PFA (see Recipes)
   
10. Block (see Recipes)
    
11. 80:20 glycerol:PBS solution (see Recipes)
    

Equipment

1. Two fine forceps (e.g. Dumont #5 tweezers)
   
2. Two deyolking tools (e.g. insect pin super glued in the end of a glass capillary tube, Figure 1)
   
3. Bench rocker
   
4. 1.5-2 ml Microcentrifuge tubes
5. Glass Pasteur pipettes
6. Pipette pump
7. Micropipettes
8. Micropipette tips
9. Microscope slides
10. Microscope slides
    
11. Dissecting microscope
    
12. Microscope for image acquisition (e.g. Zeiss Axio Imager running AxioVision software)
    
    
    Figure 1. Deyolking tools. Deyolking tools can be used to surgically remove the yolk sac from fixed zebrafish embryos. Deyolking tools consist of insect pins (Fine Science Tools, catalog number: 26002-20) super glued into the ends of glass capillary tubes (Sutter Instruments, catalog number: BF100-50-10). The glass capillary tubes are then wrapped in lab tape.
    

Procedure