Flourescent Immunostaining Protocol for a-Bungorotoxin (AChRs) in Zebrafish   

Abstract

Materials and Reagents

1. Paraformaldehyde (USB Corporation, catalog number: 19943 )
2. Phosphate buffered saline (PBS)
3. DMSO
4. Triton-100
5. Na₂HPO₄
6. NaH₂PO₄
7. Collagenase (Sigma-Aldrich, catalog number: C-9891 )
8. α-Bungorotoxin conjugated to Alexa 594 (Life Technologies, Invitrogen™, catalog number: B-13423)
9. Vectashied (Vector Lab, catalog number: H-1400 )
10. Primary antibody
    
11. Secondary antibody
12. Incubation Buffer (IB) (see Recipes)
13. 0.1 M phosphate buffer(7.4) (see Recipes)
    

Equipment

1. Rotator (Storvall Life Science, the Belly Dancer)
2. Fluorescence microscope
3. Transfer pipette
   

Procedure

1. Dechorinate embryos and remove all E3 buffer.
2. Fix the embryos using 4% paraformaldehyde in PBS+1% DMSO at RT for 4 h or O/N at 4 °C.
3. Remove paraformaldehyde with a transfer pipette. Wash 3 x 10 min with PBS (don’t use PBST).
4. Add 0.1% (1 mg/ml in 1x PBS) collagenase and incubate at RT. Treatment times vary according to age: up to 24 h=6 min; 24 to 36 h=9 min; 48 h=45 min; 3 d=75 min; 4 d=?; 5 d=Don’t collagenase treat, peel larvae using sharp forceps instead.
5. Remove collagenase solution. Wash 3 x 5 min in PBS.
6. Incubate 30 min in 10 μg/ml a-Bungorotoxin conjugated to Alexa 594 (molecular probe B-13423) in IB at RT.
   
7. Wash 3 x 5 min in IB.
   
8. If doing double staining, incubate the embryos in primary antibody diluted in IB O/N at 4 °C. Otherwise go to step 11.
9. Remove primary antibody, wash 6 x 10min in IB.
   
10. Remove IB, replace with secondary antibody diluted in IB.
    
11. Remove secondary antibody, wash 6 x 10 min in IB.
12. Remove IB, replace with 1 drop Vectashield. Let embryos settle to bottom of tube overnight. Store embryos at 4 °C.
    
13. Mount embryos in Vectashied.
    

Recipes

1. Incubation Buffer (IB) (400 ml)
   BSA                                        0.8 g
   10%Triton-100                        20 ml
   0.1M phosphate buffer (7.4)     380 ml
   Store at 4 °C
   
2. 0.1 M phosphate buffer(7.4)    400 ml
   0.2 M Na₂HPO₄            162 ml
   0.2 M NaH₂PO₄            38 ml
   H₂O                           200 ml
   

Acknowledgments

This protocol was developed in the Michael Granato Lab at University of Pennsylvania, Philadelphia, USA, and this work was supported by NIH grant R01HD037975.