BenchMasteryMolecular biology techniques & virtual lab

Immunofluorescence

Immunofluorescence on cultured cells grown on coverslips or chamber slides. The standard way to localise proteins in cell lines.

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Immunocytochemistry

Immunofluorescence on cultured cells grown on coverslips or chamber slides. The standard way to localise proteins in cell lines.

In this lesson · about 1 min read
  1. Seeing where a protein is
  2. Getting an honest image
  3. More than one colour
  4. Key terms
  5. When to use it
  6. Sub-techniques

1Seeing where a protein is

Immunofluorescence uses antibodies to label a protein inside fixed cells and a fluorescent dye to see it. Cells are fixed to freeze their structure, permeabilised so antibodies can get in, blocked to stop non-specific sticking, then incubated with a primary antibody against the target and a fluorescent secondary antibody against the primary. A DNA dye such as DAPI marks the nuclei for orientation.

PFA / methanol

Fix

PFA cross-links proteins and keeps morphology; cold methanol precipitates proteins and permeabilises at the same time.

Triton / saponin

Permeabilise

Detergent opens membranes so antibodies reach intracellular targets.

serum / BSA

Block

Occupies sticky sites; serum from the secondary's host species works best.

1° then 2°

Indirect detection

Several labelled secondaries bind each primary, amplifying the signal.

2Getting an honest image

Fluorescence images are measurements. Set the exposure so the brightest real structures sit below the camera's maximum (saturated pixels lose information), keep settings identical between conditions you compare, and always image a secondary-only control at the same settings to see what background the secondary alone gives.

3More than one colour

For two targets, use primaries from different host species (e.g. mouse and rabbit) and secondaries that recognise only one species each, with dyes whose spectra are well separated (e.g. Alexa Fluor 488 and 594). Check bleed-through with single-stained samples.

Key terms

Practise these as flashcards →
Primary / secondary antibody

The primary binds the target; the labelled secondary binds the primary's constant region.

Permeabilisation

Making membranes porous with detergent or alcohol so antibodies can enter.

Secondary-only control

Sample processed without primary; reveals non-specific secondary binding.

Saturation

Pixels at the camera's maximum value; their true intensity is unknown.

Bleed-through

Signal from one dye detected in another dye's channel.

Antifade

Mounting medium that slows photobleaching of fluorophores.

When to use it

  • Finding out where a protein sits in the cell (nucleus, mitochondria, membrane, cytoskeleton).
  • Checking whether two proteins co-localise or a treatment moves a protein.
  • Counting cells positive for a marker in a field (e.g. Ki-67 for proliferation).

Compared with alternatives

Immunohistochemistry →

The same idea in tissue sections, often with a brown chromogen instead of fluorescence.

Fluorescent proteins & dyes →

GFP fusions and live dyes label proteins in living cells without antibodies.

Confocal microscopy →

Removes out-of-focus light for sharper images and 3D stacks.

Flow cytometry & FACS →

Quantifies a marker in thousands of cells but shows no location.

Western blot →

Confirms the antibody recognises one protein of the right size.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

Immunofluorescence on cultured cells grown on coverslips or chamber slides. The standard way to localise proteins in cell lines.