Whole-mount immunostaining

Whole-mount immunostaining for dissected brains 

1. Fix whole fish overnight in 4% PFA (4%PFA, PH7.2-7.4, with 1.2% glucose and 0.34mM CaCl2) at 4 degrees

2. Quick wash with 1XPBT(0.3% triton in 1XPBS) 3 times  

3. Dissect brains in 1XPBS

4. Quick wash with 1XPBT once before adding 100-150ul blocking buffer (2%BSA in PBS+0.3% triton), block for 1 hour at 4 degrees

5. Add primary antibodies diluted accordingly in blocking buffer and shake gently on shaker at 4 degrees (duration depends on antibodies, 2 days work better for PERK).  

6. Wash with PBT 3 times (10 minutes each)

7. Block with blocking buffer for 1 hour at 4 degrees

8. Add secondary antibodies (diluted in blocking buffer) for 1-2 days at 4 degrees on shaker

9. Wash 3X with PBT

10. Add 70% glycerol in 1XPBS and mount on slides, ventral side up

11. Image with confocal microscope