Immunofluorescence microscopy that can be interpreted: choosing fixation and permeabilisation for the epitope, picking an immunofluorescence antibody, an antibody immunofluorescence pairing and an immunofluorescence secondary antibody or secondary antibody immunofluorescence layer that cannot collide with anything else on the slide, what multiplex immunofluorescence adds in panel design, spectral separation and the image analysis microscopy burden that follows, and the controls that decide what the image means

Building a protocol that can be trusted

1. Choose fixation for the epitope and the structure. Aldehyde fixation preserves structure by cross-linking and can mask epitopes, which is what retrieval exists to reverse. Alcohol fixation precipitates protein, preserves some epitopes better and disturbs membranes and cytoskeleton. Test both on a known positive rather than inheriting a protocol.
2. Permeabilise according to where the target is. A surface antigen may need no permeabilisation at all, and permeabilising it can move the signal. A nuclear target needs a stronger detergent than a cytoplasmic one. Match the agent and the time to the compartment.
3. Block against the actual source of background. Serum from the species the secondary was raised in blocks non-specific binding; a protein block handles general stickiness; and for tissue with endogenous biotin or strong autofluorescence, neither helps and a specific treatment is required.
4. Choose secondaries that cannot collide. Cross-adsorbed secondaries raised in one host against different primary species are the clean way to multiplex. Where two primaries share a host, sequential detection with a blocking step in between is the alternative, and it needs its own control.
5. Run the controls on the same slide and the same settings. A no-primary control, a known positive and, where possible, a genetic negative. Acquire all of them at the settings used for the experiment, because a control imaged at a lower exposure proves nothing at all.

Autofluorescence and how to deal with it

Fixed tissue, red blood cells, lipofuscin and aldehyde fixation itself all fluoresce, mostly in the green, which is where the brightest conjugates sit. Move the target to a longer wavelength before reaching for a quenching reagent.

Image an unstained section at the experiment's settings before staining anything. It tells you immediately how much of what you are about to see was already there.

Mounting, storage and fading

An antifade mountant and dark storage at four degrees are the minimum. Measure the fade rate on your own combination rather than trusting the datasheet, because it depends on the fluorophore, the mountant and the illumination you use.

Seal the coverslip if slides will be re-imaged. Mountant that dries at the edge pulls the coverslip and moves the focal plane, which looks like sample drift.

From an image to a figure

Keep the raw acquisition files and do the analysis on them. Adjusting brightness for a figure is acceptable if it is applied equally to every panel including the controls, and stated; anything else is not.

Include the control panel in the figure. A published immunofluorescence image without its no-primary control is asking the reader to take the hardest part on trust.

Common questions

Which fixation should I start with for immunofluorescence microscopy?

Try paraformaldehyde and a cold alcohol fixation side by side on a known positive sample. Which one works is a property of the epitope, and inheriting another laboratory's choice is the commonest reason a published antibody does not work for you.

How do I choose an immunofluorescence secondary antibody?

Raised against the host of your primary, not raised in your sample's species, cross-adsorbed where species could collide, and conjugated to a fluorophore that suits your filters and the target's abundance. Titrate it like a primary.

What causes high background?

An over-concentrated secondary, inadequate blocking, endogenous autofluorescence in the tissue, or drying of the sample at any stage. Run the secondary-only control first: it separates the secondary from everything else in one experiment.

Can I quantify an immunofluorescence image?

Only with fixed acquisition settings, a control on every slide and a pre-declared analysis. Auto-exposure across a cohort destroys quantification, and thresholds chosen after seeing the images are not measurements.

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