Table 1 Recommendations on working with DSP stock and working dilution, as described by Attar et al. [16]

Preparation of 50× DSP stock

• Equilibrate DSP vial at RT for 30 min and then prepare a 50× stock solution of DSP (50 mg/mL) in anhydrous dimethyl sulfoxide (Sigma, Cat. N. 276855-100ML).

• Dispense the stock into single-use aliquots (e.g., 100 μL aliquots, but volume depends on your use) and store in a bag and dry environment (with silica/desiccant if possible) at −80 °C.

• Be mindful of not opening and closing the tubes at −80 °C.

Preparation of DSP working dilution

• Thaw the 50× DSP stock reagent from −80 °C and equilibrate at RT no longer than 10 min before fixation.

• Immediately before use prepare 500 μL of 1× DSP working solution in molecular biology grade 1× PBS as follows: aliquot 10 μL of 50× DSP stock reagent in a 1.5-mL tube and while vortexing (VERY IMPORTANT) add 490 μL of PBS dropwise using a P200 pipette. 1× DSP must be used within 5 min of preparation.

• Note: Do not prepare larger volumes (e.g., if you need to fix two samples, prepare each aliquot of 500 μL separately, DO NOT prepare 1 mL and then aliquot into two tubes). You should notice some thin white rings on the walls of the tube once diluted the 50× stock. This is expected and will be cleared during filtration. Stronger precipitation indicates insufficient solving of DSP and preparation of a new dilution is strongly recommended.

• Filter the 1× DSP working dilution using a 40-μm Flowmi strainer (Sigma, cat. no. BAH136800040-50EA). After filtration 1× DSP can be pooled.

• Do not re-freeze the leftovers of the 50× DSP. Always use a freshly thawed aliquot to prepare the 1× working solution.