Protocols that we’ve collected from collaborators and conversations that aren’t currently our standard operating procedure, but are excellent and we don’t want to forget.

Table of Contents

Alternative mounting medium

Hiura Lab shared that they have concerns about auto-fluorescence from mounting solutions that have DAPI in them, so use this method instead. This is from their protocol.

DAPI Cat #: D9542-1MG from MilliporeSigma. Cas: 28718-90-3

Mowiol Mounting Medium Recipe

Needed:

• Mowiol 4-88 (Cat #: 81381-50G from Sigma-Aldrich. Cas: 9002-89-5. (Also called Polyvinyl alcohol 4-88).
• Glycerol
• Tris-Cl (0.2 M, pH 8.5)
• DABCO (1,4-diazabicyclo-[2,2,2]-octane)

To do:

Add 2.4 g of Mowiol 4-88 to 6 g of glycerol. Stir to mix. Add 6 mL of H2O and leave for several hours at room temperature. Add 12 mL of 0.2 M Tris-Cl (pH 8.5) and heat to 50°C for 10 min with occasional mixing. After the Mowiol dissolves, clarify by centrifugation at 5000g for 15 min. For fluorescence detection, add DABCO to 2.5% to reduce fading. Aliquot in airtight containers and store at −20°C. Stock is stable at room temperature for several weeks after thawing.