Technologies
Transmission Electron Microscopy (TEM)
Passes an electron beam through an ultra-thin specimen to image internal ultrastructure with atomic-level resolution.
Brightfield Microscopy (BFM)
Basic trans-illumination where contrast arises from light absorption by the specimen; typically used for fixed, stained tissues.
Description
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.