Immunofluorescence microscopy: fixation, blocking and the controls that make it interpretable

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.

Where multiplex immunofluorescence changes the panel

Beyond three or four colours the problem stops being the microscope and becomes the chemistry: the secondary antibodies have to come from species that do not cross react, the fluorophores have to be separable on the filters you own, and every additional marker narrows what is left for the next one. Cyclic methods get round that by stripping and restaining, at the cost of a section that has to survive several rounds and a registration step between them.

Whichever route, the control that matters is the panel MINUS one antibody, repeated for each: it is the only way to show that a signal is the marker rather than bleed through or an unblocked secondary. Budget those controls into the section count before the experiment, because they cannot be added afterwards to a section that is gone.

An apex2 antibody and proximity labelling

APEX2 is an engineered peroxidase fused to a bait so that nearby proteins are biotinylated in living cells, so an apex2 antibody confirms the fusion is expressed and localised while the experiment's readout is the biotinylated proteome. Expression level matters because overexpression mislocalises the bait, which is why the antibody and an imaging check come before the labelling is trusted.

A titin antibody and the largest protein in the cell

Titin spans half a sarcomere and is far too large for a standard gel, so a titin antibody is used in imaging, where its epitope's position along the molecule gives a striated pattern that itself reports sarcomere structure. On a gel it needs a very low percentage or an agarose system, and a smeared high mass band is the usual sign the separation was wrong.

A gli2 antibody and a factor processed by the pathway

GLI2 is proteolytically processed into a repressor form unless the hedgehog pathway is active, so a gli2 antibody has to say which form it detects and both bands belong in a figure claiming pathway activity. It is nuclear when active, so an image with a nuclear counterstain carries information a lysate does not. A pathway agonist treated control is what produces the full length species.

A rara antibody and a receptor that moves on ligand

The retinoic acid receptor alpha is nuclear whether or not ligand is present, so a rara antibody reports expression while the pathway is read as target gene induction, and a fusion protein in leukaemia is a separate clinical question with its own reagents. The family shares a domain, so specificity against the beta and gamma receptors belongs in the datasheet.

A sox11 antibody and a developmental factor re-expressed in tumours

SOX11 is a developmental transcription factor used in pathology because it is re-expressed in particular lymphomas, so a sox11 antibody is scored as nuclear positivity with a validated clone and a stated cutoff. The SOX family shares a DNA binding domain, so cross reactivity against the closest members is what decides whether a call is safe.

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 with an immunofluorescence antibody?

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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