Fluorescence microscopy: c-Fos immunofluorescence

A fluorescence microscope shows where particular molecules are in a cell. Each fluorescent dye (a fluorophore) absorbs light of one colour and emits light of a longer wavelength. The microscope shines a narrow band of excitation light on the specimen and lets only the emitted light reach the camera, using filters and a dichroic mirror (Lichtman & Conchello, 2005). With dyes of different colours, imaged one wavelength band at a time, several markers can be seen in the same neurons: DAPI labels every nucleus in blue, NeuN labels the nuclei of most neurons (Mullen et al., 1992), a fluorescent Nissl stain labels neuronal cell bodies, and an antibody against c-Fos labels recently activated neurons.

When a neuron is strongly activated, it switches on a set of genes within minutes. One of these immediate-early genes is c-fos; its protein product, c-Fos, accumulates in the cell nucleus over the following hours and then disappears again, so staining for c-Fos gives a snapshot of which neurons were recently active (Sagar et al., 1988; Morgan & Curran, 1991). Psychostimulants such as amphetamine strongly induce c-fos in the striatum, shown in rats by Graybiel et al. (1990); noxious stimulation of the skin induces c-Fos in the superficial dorsal horn of the spinal cord (Hunt et al., 1987, in rats), and pressing a sensitised hind paw does so on the same side in mice (Sharma et al., 2009). c-Fos immunoreactivity is also studied in post-mortem human tissue, where groups are defined by diagnosis or by the circumstances of death (Nogami et al., 1999; Patodia et al., 2021).

Immunohistochemistry uses antibodies to find one protein among thousands. A primary antibody raised in one species binds the target; a secondary antibody raised against the antibodies of that species carries the dye. Human tissue brings its own problems: it is fixed by immersion and often stored for a long time in formalin, which masks epitopes (Evers & Uylings, 1994; Lyck et al., 2008), and aged neurons contain lipofuscin, whose autofluorescence can be quenched with Sudan Black B (Romijn et al., 1999).

The sections are simulated inside real atlases: the Allen Mouse Brain Common Coordinate Framework (Wang et al., 2020; image credit: Allen Institute), the Waxholm Space atlas of the Sprague Dawley rat brain (Papp et al., 2014; Kleven et al., 2023; CC BY 4.0), the Allen Reference Atlas – Mouse Spinal Cord (Allen Institute for Brain Science, 2009; drawings by Charles Watson and Gulgun Sengul; image credit: Allen Institute) and BigBrain, a human brain cut into 7,404 cell-body-stained sections (Amunts et al., 2013; CC BY-NC-SA 4.0). Human spinal cord is not offered, because the only human spinal cord atlas available is an MRI template without cell-level histology.

Learning objectives

• Explain how a fluorescence microscope separates markers by wavelength: excitation and emission spectra, the Stokes shift, filter cubes, dichroic mirrors and bleed-through
• Operate an epifluorescence microscope: objective, numerical aperture, focus, exposure, saturation and photobleaching, and acquire one image per channel
• Find a target region (striatum, superficial dorsal horn or neocortex) by comparing sections with a real reference atlas
• Design a multi-label immunofluorescence panel: match secondary antibodies to the host species of the primaries, avoid secondaries against the tissue's own species, choose dyes that do not overlap, and include a no-primary control
• Explain what c-Fos shows, why timing matters, and how human post-mortem tissue differs: consent, matching, post-mortem interval, fixation, antigen retrieval and lipofuscin
• Count c-Fos-positive nuclei with a threshold set from the background (background + k × SD), validate it by eye, test its robustness, and compare groups with a Welch t-test, confidence interval and effect size

Ethics and safety

Work with animals needs approval by an Institutional Animal Ethics Committee (in India, the CCSEA under the Prevention of Cruelty to Animals Act 1960; elsewhere, frameworks such as EU Directive 2010/63/EU, the US Animal Welfare Act and PHS Policy, and the UK Animals (Scientific Procedures) Act 1986), trained and authorised staff, and an ethical review that applies the 3Rs: replace animals where possible, reduce their number with a good design and a sample-size calculation, and refine procedures to minimise suffering. A noxious stimulus needs a strong justification and the mildest effective stimulus. Perfusion is carried out under deep terminal anaesthesia, after the absence of reflexes has been confirmed. Report animal studies following the ARRIVE guidelines, developed by the NC3Rs in the United Kingdom and endorsed by journals internationally (Percie du Sert et al., 2020). Human tissue comes only from consented donation through a brain bank, under the rules of the country (in India, the ICMR National Ethical Guidelines of 2017; in England, Wales and Northern Ireland, the Human Tissue Act 2004), and is used only for projects with ethics committee approval; donors are coded and nothing is done to them for the study. Paraformaldehyde and formalin are toxic and are used in a fume hood with gloves and eye protection; amphetamine is a controlled drug that needs licensed storage and records.

Key terms

• c-Fos: The protein made from the immediate-early gene c-fos; it builds up in the nucleus of a neuron for some hours after strong activation and is used as a marker of recent activity.
• Primary antibody: An antibody that binds the target protein in the tissue; it is made in a host species such as rabbit, mouse, rat, goat or chicken.
• Secondary antibody: An antibody that binds the primary antibodies of one host species (for example donkey anti-rabbit) and carries a fluorescent dye.
• Fluorophore: A molecule that absorbs light at one wavelength and emits light at a longer wavelength, for example DAPI or Alexa Fluor 488.
• Stokes shift: The difference between the wavelength a fluorophore absorbs best and the wavelength it emits best; it lets filters separate the emitted light from the excitation light.
• Filter cube: The set of an excitation filter, a dichroic mirror and an emission filter that selects which dye is excited and which emitted light reaches the camera.
• Dichroic mirror: A mirror that reflects light below a cut-off wavelength and transmits light above it, sending excitation light to the specimen and emitted light to the camera.
• Bleed-through: Signal from one dye that appears in the channel meant for another dye because their spectra overlap.
• Numerical aperture (NA): A number describing how wide a cone of light an objective collects; a higher NA gives finer resolution and, at the same magnification, a brighter fluorescence image.
• Photobleaching: The gradual, permanent loss of fluorescence of a dye while it is illuminated.
• Saturation: A pixel that has reached the highest value the camera can record; brighter parts of the specimen can no longer be told apart and intensities cannot be measured.
• DAPI: A blue fluorescent dye that binds DNA and labels the nucleus of every cell.
• Bregma: A landmark on the skull used to give positions in the brain; coronal sections are named by their distance in front of or behind it.
• Unbiased counting frame: A square sampling frame with two inclusion and two exclusion edges, so that each cell is counted once and cells cut by the frame are not counted twice.
• Brain bank: An organisation that collects brains donated with consent after death, records the clinical and post-mortem details, fixes or freezes the tissue and releases coded samples to approved research projects.
• Post-mortem interval: The time between death and the fixation or freezing of the tissue.
• Antigen retrieval: A treatment, usually heating in a buffer, that partly reverses the masking of epitopes by formalin cross-links so that antibodies can bind again.
• Lipofuscin: Granules of oxidised lipid and protein that build up in ageing neurons and glia and fluoresce over a broad range of wavelengths.
• Superficial dorsal horn: Laminae I and II of the spinal grey matter, where most fine sensory fibres that signal painful stimuli end.
• Threshold (image analysis): The intensity above which a pixel is treated as part of a labelled object; here set as the local background plus k times the standard deviation of the background.