Fixing and labelling cells for immunofluorescence (IF) microscopy

	Protocol & Procedures Document
Protocol Name	Fixing and labelling cells for immunofluorescence (IF) microscopy
Materials Required	4% Paraformaldehyde Blocking buffer (0.5% BSA/PBS, or equivalent) Pemeabilisation buffer (0.1% Triton X-100) PBS Parafilm Labels (primary antibodies &/or fluorescently labelled secondary antibodies &/or Fluorescent markers (e.g. Phalloidin, Cell mask, etc.) Mounting media (IMBiol, Prolong Diamond, etc.) Sealant (clear nail polish)
Procedure	Fixation: Fix cells with 4% Paraformaldehyde for at least 30 minutes Wash fixative off cells thrice with PBS Permeabilisation: If required (antibody labelling/internal labelling) permeablise with 0.1% Triton X-100 for no more than 5 minutes. Wash cells thrice with PBS Blocking: Block with 0.5% BSA/PBS for at least 30 minutes to prevent non-specific antibody labelling, and to reduce background labelling. Primary antibody labelling: Prepare primary antibodies in 0.5% BSA/PBS as per manufacturer’s instructions. Add antibody mixture to cells for at least 45 minutes Wash cells thrice with BSA/PBS Secondary antibody labelling: Prepare secondary antibodies in 0.5% BSA/PBS as per manufacturer’s instructions. Add antibody mixture to cells for at least 30 minutes Wash cells thrice with 0.5% BSA/PBS Wash coverslip once with PBS 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. Cleaning slide: Always clean both slide and coverslip.
Notes	The inverted method for labelling coverslips is used ideally to minimise the amount of labelling reagents required Labels such as Alexa dyes, JFs, or SiR will last longer than expressed protein labels such as GFP/mCherry Imaging freshly prepared coverslips will always yield better results. Number 1.5 coverslips are best and are available in the IMB store (Cat# D766)
Version	V1 – April 2018