Arnolds' silver staining

Living Embryos

1. In a culture dish, briefly wash embryos in ddH₂O several times, and then incubate the embryo in ddH₂O for at least 5 min to remove excess ions, especially Cl−.
2. Replace ddH₂O with sodium borate solution (30 mM, pH 7.5) and incubate for 1 min.
3. Replace sodium borate solution with freshly prepared silver methenamine solution and incubate briefly.
4. Place the dish under a dissecting microscope, and direct fiber optic illumination onto the embryos at the highest intensity. Two illuminators can be used in this step. Watch the reaction closely. Cell outlines should turn dark brown within 10-15 min. Terminate the reaction by turning off the illumination and replacing the silver methenamine solution with sodium borate solution (30 mM, pH 7.5).
5. Fix the embryos in 4% formaldehyde in 0.25X PBS or other buffered solution.

Fixed Embryos

1. Fix embryos in 0.8% formaldehyde in sodium cacodylate buffer (100 mM, pH 7.4) for 30 min or less at room temperature. Overfixation prevents visualization of the epithelial junctions.
2. Wash briefly the embryos several times with ddH₂O.
3. Incubate the embryos in ddH₂O for 5 min.
4. Replace ddH₂O with sodium borate solution and incubate for 1 min.
5. Replace sodium borate solution (30 mM, pH 7.5) with freshly prepared silver methenamine solution and incubate briefly.
6. Place the dish under a dissecting microscope, and direct fiber optic illumination onto the embryos at the highest intensity. Two illuminators can be used in this step. Watch the reaction closely. Cell outlines should turn dark brown within 10-15 min. Terminate the reaction by turning off the illumination and replacing the silver methenamine solution with sodium borate solution (30 mM, pH 7.5).
7. Fix the embryos overnight at 4ºC in Carnoy’s fixative and store the embryos in ethanol.

Recipes

• Silver methenamine solution: Combine 1 part of 0.3% AgNO3, 1 part of 3% hexamethylenetetramine, and 1 part of 90 mM sodium borate (pH 7.5). Prepare immediately before use. Remove particles in the silver methenamine by filtration if necessary.