Anisomycin Dilution

This protocol is used for the dilution of anisomycin (Sigma A9789) for intracranial infusion (10 mg/mL). Notes for diluting higher concentrations are also included.

Stock Solutions:

0.1 N HCL: Take 1N HCL and dilute in 1X PBS to 0.1N HCL. This will keep final salt concentration close to the vehicle (1X PBS).

Instructions for diluting to 10 mg/mL:

1. Weigh out 5-10 mg anisomycin in a 1.5 mL Eppendorf tube.
2. Add 0.1N HCL solution to anisomycin, such that it is half the final volume, and vortex. For example, if dissolving 10 mg anisomycin to 10 mg/mL, final volume will be 1 mL, and we’d add 500 uL of the 0.1 N HCL solution.
3. Add 1X PBS in order to bring to 90% of final volume. Going back to prior example, if dissolving 10 mg anisomycin to 10 mg/mL, we’d add 400 uL of PBS (500 0.1N HCL + 400 PBS = 900)
4. In order to normalize pH, slowly add 1N NaOH in 1-2 ul increments until pH is close to 7 (6-8). When diluting 10 mg, this usually takes about 5-10 ul NaOH.
   *** Because we usually are making small volumes that aren’t compatible with using a pH meter, we pipet small amounts (0.5 uL) onto pH test strips. Be sure to briefly vortex after each addition of NaOH to allow pH to equilibrate.
5. Bring to final volume with 1X PBS

For diluting to higher concentrations.

For higher final concentrations of anisomycin (e.g., 150 mg/mL), we modify step 2 above such that initial volume of HCL solution added is 80-90% of final volume, skip step 3, and then adding more NaOH to balance the pH (1 ul of 1N NaOH should approximately balance 10 ul 0.1 N HCL).