Immunolocalization for Light and Electron Microscopy

This training activity provides theoretical and hands-on instruction in the localization of specific proteins, antigens or other cellular components using antibody-based techniques for light and electron microscopy.

The training covers the main stages of immunolocalization, from sample preparation and antibody selection to image acquisition and interpretation.

Immunolocalization for Light Microscopy

Light microscopy immunolocalization techniques allow the detection and spatial localization of target molecules in cells and tissues using fluorescent or enzyme-conjugated antibodies.

The training includes:

• Selection and preparation of biological samples

• Fixation methods compatible with antigen preservation

• Paraffin, frozen or resin section preparation

• Deparaffinization and rehydration when required

• Antigen retrieval procedures

• Permeabilization and blocking

• Selection and dilution of primary and secondary antibodies

• Direct and indirect immunofluorescence

• Enzyme-based immunohistochemical detection

• Nuclear and structural counterstaining

• Mounting and preservation of labelled samples

• Image acquisition using light, fluorescence or confocal microscopy

Immunolocalization for Electron Microscopy

Electron microscopy immunolocalization enables the detection of target molecules at the ultrastructural level, generally using antibodies conjugated to colloidal gold particles.

The training covers:

• Selection of fixation and embedding procedures compatible with antigenicity

• Use of acrylic resins suitable for immunolabelling

• Preparation of ultrathin sections

• Collection of sections on TEM grids

• Blocking and incubation procedures

• Primary antibody incubation

• Immunogold detection using gold-conjugated secondary antibodies

• Single and multiple immunogold labelling

• Post-staining of labelled sections

• Visualization and interpretation of gold particle distribution

• Quantitative analysis of immunogold labelling

Experimental Controls and Validation

Special attention is paid to the experimental controls required to verify the specificity and reliability of the labelling, including:

• Negative controls without primary antibody

• Isotype controls

• Pre-immune serum controls

• Positive control samples

• Antibody specificity assessment

• Evaluation of non-specific background

• Replication and quantitative analysis

At the end of the activity, participants will understand the main immunolocalization workflows, select suitable sample-preparation and detection methods, optimize antibody conditions and distinguish specific labelling from background or technical artefacts.