Materials and Reagents

1. Live coral
   
2. Electron microscopy grade paraformaldehyde (16% solution in 10 ml ampules) (Electron Microscopy Sciences, catalog number: 15700 or 15710 )
   
3. Ethyl alcohol (absolute, 200 proof)
   
4. Ethyl alcohol (190 proof)
   
5. Xylenes (any reagent ≥ 98.5% xylenes, other tissue clearing agents such as Safeclear, are also acceptable) (Safeclear, catalog number: 044-192 )
   
6. Paraffin embedding medium (e.g. Paraplast X-TRA) (McCormick Scientific, catalog number: 39503002 )
   
7. Triton-X-100 (Bio-Rad Laboratories, AbD Serotec^®, catalog number: 1610407 or Sigma-Aldrich, catalog number: T8787 )
   
8. Normal goat serum (Vector laboratories, catalog number: S-1000 )
   
9. Keyhole Limpet Hemocyanin (Sigma-Aldrich, catalog number: 7017 )
   
10. Phosphate buffered saline (PBS) (e.g. 10x) (Corning Incorporated, catalog number: 46-013-CM )
    
11. Microscope slides and coverslips
    
12. Custom, antigen-affinity purified, rabbit polyclonal anti-VHA_B antibodies developed using a peptide antigen matching a conserved region of the VHA_B subunit (AREEVPGRRGFPGY) (Genscript USA, Inc.). The epitope is 100% conserved among diverse animal species, and specifically recognizes VHA_B from coral (Barott et al., 2015), bone-eating worms (Tresguerres et al., 2013), hagfish (Clifford, et al. 2015), and sharks (Tresguerres et al., 2010; Roa et al., 2014).
    Note: This protocol may be adapted for other proteins following validation of the primary antibodies in the coral species to be examined. However, successful localization using different primary antibodies may require optimization of the fixation procedure from that described below.
    
13. Secondary antibodies: Goat anti-rabbit IgG Alexa Fluor 555 (2 mg/ml) (Life Technologies, catalog number: A21429 )
    Note: The Alexa555 fluorophore is used to avoid overlap with the coral’s endogenous green fluorescent protein (GFP), but other fluorophores outside of the GFP emission range may also work.
    
14. Hoescht 33342 (Life Technologies, catalog number: H1399 )
    
15. Mounting medium (e.g. Electron Microscopy Sciences, catalog number: 17895 )
    Note: Unless otherwise noted, reagents can be obtained from any general laboratory reagent manufacturer or supplier.
    
16. S22 buffer (see Recipes)
    
17. Ca-free S22 buffer + 0.5 M EDTA (see Recipes)
    
18. Fixative solution (see Recipes)
    
19. Blocking buffer (see Recipes)

Equipment

1. Chemical fume hood
   
2. Fluorescence microscope (e.g. Zeiss AxioObserver Z1, or any other microscope with the ability to detect blue (“DAPI filter”, peak excitation 350 nm; peak emission 461 nm; for Hoescht staining of nuclei) and red (“TRITC filter”, peak excitation 550 nm; peak emission 570 nm; for secondary antibodies) fluorescence.
   
3. Optional
   
   1. Coral endogenous GFP and chlorophyll from dinoflagellate symbionts (Symbiodinum) are visible using settings for “green fluorescence” (“FITC filter”, peak excitation 490 nm; peak emission 525 nm).
      
   2. Structured illumination (Zeiss Apotome2), confocal capabilities *suggested but not required.
      
   3. Differential Interference Contrast (DIC) (Nomarski microscopy) *suggested but not required.
4. Microcentrifuge [any model capable of spinning 1.5 ml tubes at 3,000 x g at 4 ℃
   Note: This temperature can be achieved by placing the microcentrifuge inside of a fridge.
   
5. Tissue cassettes (e.g. C&A Scientific, catalog number: EC-0109x )
   
6. Tissue embedding station or any other method to pour liquid paraffin (e.g. Kedee, model: KD-BM II )
   
7. Embedding base molds (metal) (e.g. C&A Scientific, catalog number: HB-07 , 15 , 24 , 30 )
   
8. Embedding rings (plastic) (e.g. Biologix, catalog number: 41-3004 )
   
9. Microtome (any model capable of cutting 5-30 μm paraffin sections)
   
10. Slide warmer/hot plate (any model capable of warming up slides to 30 ℃)
    
11. Hydrobarrier pen (Pap pen) (e.g. Vector Laboratories, catalog number: H-4000 )
    
12. Rotating platform (any rotator/shaker/rocker)

Software

1. Imaging software (e.g. Zeiss Axiovision software, Adobe Photoshop, ImageJ)

Procedure