Immunofluorescence Application Guide

How immunofluorescence works

A primary antibody binds the target antigen and fluorescence is detected directly from a conjugated primary antibody or indirectly through a fluorescent secondary antibody. Fixation, permeabilisation and imaging settings can strongly affect the result.

1. Choose the specimen format, fixation and permeabilisation strategy.
2. Block non-specific binding and optimise antibody concentrations.
3. Apply primary and detection antibodies with matched controls.
4. Acquire images consistently and assess localisation, background and spectral overlap.

Experimental planning and controls

Use a sample expected to express the target where possible and include controls that help distinguish target-dependent signal from secondary-antibody, autofluorescence or fixation-related background. A visually plausible pattern is supportive evidence but does not by itself establish specificity.