All fixation steps must be performed in a fume hood wearing the appropriate personal protective equipment (PPE). The Material Safety Data Sheet (MSDS) for each chemical must be read before commencing.
Adherent cells grown in a petri dish must be submerged in solution at all times to prevent desiccation. Petri dishes should be clearly labelled on the bottom to prevent sample mix up.
Different fixatives can influence the antigenicity of cells and tissue. The following two fixation solutions are typically tested to determine the best immunogold labelling outcome:
2% Paraformaldehyde, 0.2% glutaraldehyde in 0.1 M phosphate buffer:
Remove cells from the incubator and gently add a volume of double concentrate fixative, 4% paraformaldehyde, 0.4% glutaraldehyde in 0.1 M phosphate buffer (PB) equal to the volume of culture media in the petri dish. Ensure the fixative and culture media are well mixed, and incubate for 5 mins at room temperature.
4% Paraformaldehyde in 0.1 M phosphate buffer:
Remove cells from the incubator and gently add a volume of normal concentrate fixative, 4% paraformaldehyde in 0.1 M PB equal to the volume of culture media in the petri dish. Ensure the fixative and culture media are well mixed, and incubate for 5 mins at room temperature. Double strength fixative is not used with this fixation solution to avoid osmotic shock of cells.