X-34

Supplied Reagents:

1. X-34: Create a 5 mM stock solution of X-34 (200X) using the X-34 diluent. Create a 25 µM staining solution by further diluting the 5 mM stock solution using the X-34 diluent.
Note: X-34 diluent (40% EtOH, pH 10) is the recommended solvent for staining. A slight precipitate may be present, but will not interfere with the product's performance.

Additional Reagants (Not Supplied)

1. X-34 diluent (40% EtOH, pH 10): Combine 20 mL of EtOH in 29.5 mL of dH₂O. Add 1 µL of 5M NaOH dropwise until the solution reaches pH 10. Bring the final volume of X-34 diluent to 50 mL with dH₂O.
2. Differentiation buffer: 50 mM NaOH, 80% EtOH

Protocol:
1. Stain slides in 25 µM X-34 staining solution for 30 min at room temperature in the dark.
2. Rinse slides three times in dH₂O.
3. Incubate for 2 min in the differentiation buffer.
4. Rinse slides three times in dH₂O.
5. If desired, proceed with immunostaining.
6. Mount and image.

Store lyophilized at 4ºC, desiccated. In lyophilized form, the product is stable for 12 months.

X-34 is a lipophilic and highly fluorescent marker used to examine pathological changes in Alzheimer’s disease (AD). This cellular dye was developed as a derivative of Congo red to be more compatible with in vivo applications. By replacing the azo groups (nitrogen-to-nitrogen double bonds) in Congo red with carbon-to-carbon double bonds and having more lipophilic salicylic acid groups, X-34 has proven to be highly fluorescent and have increased membrane permeability. X-34 detects β-sheet structures and labels amyloid plaques, neurofibrillary tangles (NFTs), neuropil threads, and vascular amyloid in AD brains (1).