X34 staining

X34 staining to detect amyloid structures in C. elegans:

1. Dissolve X-34 from Sigma (SML1954) into DMSO at 25 mg/ml. From this stock solution, make 1 mM X-34 in 10 mM Tris-HCl (pH 8).

1. Place 50 adult C. elegans of the same age into an Eppendorf tube with 1 ml M9 with 0.01% Triton.

2. Vortex briefly at low speed and spin down in mini-centrifuge at low speed. Remove supernatant. If bacteria still present, wash again with 1ml M9 with 0.01% Triton.

3. Remove supernatant and add 100 μl of 1 mM X-34. Place on nutator (in the dark) and gently shake at room temperature for two hours.

4. Spin down in mini-centrifuge at low speed and remove supernatant. To worm pellet, add 100 μl M9 with 0.01% Triton. Pipette worms onto seeded NG plate (cut end of pipette tip to enlarge opening). Let dry and leave the worms to destain overnight.

5. Next day, prepare fresh 2% agarose pad and pick destained worms into 15 μl M9 with 2 µM levamisole for anaesthesia.

6. Proceed to imaging with confocal microscope using 405 nm for excitation and an emission range between 470-520 nm.