Institute for Molecular Bioscience

Fixing and labelling cells with Wheat Germ Agglutinin (WGA)

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Protocol & Procedures Document 

Protocol Name 

Fixing and labelling cells with Wheat Germ Agglutinin (WGA) 

Materials Required 

  • 4% Paraformaldehyde 

  • PBS 

  • Parafilm 

  • Labels (eg. Alexa488/555/647) 

  • Mounting media (IMBiol, Prolong Diamond, etc.) 

  • Sealant (clear nail polish) 

Procedure 

  1. Fixation: Fix cells with 4% Paraformaldehyde for at least 30 minutes 

  2. Wash fixative off cells thrice with PBS 

  3. Fluorescence labelling: Prepare WGA* in PBS and add mixture to cells for at least 45 minutes 

  4. Wash cells thrice with PBS 

  5. Wash coverslip once with Water** 

  6. Coverslip mounting: Add a drop of mounting media onto a clean glass slide***. Place dry coverslip cells side down onto mounting media. Seal the slide with a sealant or let the mounting media cure for at least 60 minutes. 

  7. Cleaning slide: Always clean both slide and coverslip after nail polish has dried 

Notes 

  • The inverted method for labelling coverslips is used ideally to minimise the amount of labelling reagents required 

  • Imaging freshly prepared coverslips will always yield better results. 

  • Number 1.5 coverslips are best and are available in the IMB store (Cat# D766) 

  • * = Generally speaking the following concentrations work for WGA (A350 1:50, A488 1:500, A555 1:200, A647 1:400 TMR 1:500) 

  • **A quick wash in water prevents PBS crystals forming 

  • ***Clean the slide by giving it a quick wipe with EtOH using a Kimwipe

Version 

V1 – April 2019