M. Others at CTEM - Servicio de Microscopía Confocal y Electrónica de Transmisión de la EEZ. CSIC

Vibratome sectioning is a technique used to obtain relatively thick sections of fresh, fixed or lightly processed biological samples without the need for paraffin or resin embedding.

A vibrating blade moves through the specimen while the sample is supported in a liquid-filled chamber. This reduces mechanical compression and helps preserve tissue architecture, cellular organization and antigenicity.

Applications

Vibratome sectioning is particularly suitable for (among others):

Brain and nervous tissue

Animal organs and soft tissues

Plant tissues and organs

Thick tissue slices for fluorescence microscopy

Immunohistochemistry and immunofluorescence

In situ hybridization

Confocal laser scanning microscopy

Correlative light and electron microscopy

Selection of specific regions for subsequent electron microscopy processing

Sample Preparation

Samples may be processed fresh or after chemical fixation, depending on the experimental objective. The specimen is usually attached to a sample holder and immersed in an appropriate buffer or physiological solution during sectioning.

Section thickness can be adjusted according to the sample type and downstream application. Vibratome sections are generally thicker than ultramicrotome sections and commonly range from approximately 30 to several hundred micrometres.

Advantages

Vibratome sectioning offers several advantages:

No paraffin or resin embedding is required

Reduced exposure to heat and organic solvents

Good preservation of tissue morphology

Improved preservation of fluorescent proteins and antigens

Suitable for relatively large or fragile samples

Production of free-floating sections for staining and immunolabelling

Possibility of obtaining viable tissue slices under appropriate conditions

Limitations

The quality of vibratome sections depends on tissue consistency, fixation, blade condition, cutting speed, vibration amplitude and section thickness.

Very soft, fibrous or highly heterogeneous samples may require protocol optimization or additional support.

Cryostat Sectioning

Cryostat sectioning is used to obtain thin sections of frozen biological specimens for examination by light, fluorescence or confocal microscopy. The Leica Jung Frigocut 2800 combines a microtome with a refrigerated chamber, allowing samples to remain frozen throughout the sectioning process.

Fresh or previously fixed specimens are rapidly frozen and mounted on a specimen holder using an appropriate cryo-embedding medium. Sections of the required thickness are then cut inside the refrigerated chamber and collected on microscope slides for subsequent staining, labelling or imaging.

Applications

Cryostat sectioning is suitable for (among others):

• Histological examination of frozen tissues

• Immunohistochemistry and immunofluorescence

• Localization of proteins and other molecular targets

• Fluorescence and confocal microscopy

• In situ hybridization

• Enzyme histochemistry

• Analysis of tissues containing lipids or other components that may be altered during conventional processing

• Rapid evaluation of tissue morphology

• Identification of regions of interest for subsequent microscopy studies

• Morphometric and quantitative image analysis

Types of Samples

The technique may be applied to (among others):

• Animal tissues and organs

• Plant tissues

• Cell aggregates and organoids

• Experimental tissue models

• Fresh-frozen specimens

• Fixed and cryoprotected specimens

The suitability of a sample depends on its size, consistency, freezing method and intended downstream application.

Advantages

Cryostat sectioning provides several advantages:

• No paraffin or resin embedding is required

• Reduced exposure to heat and organic solvents

• Rapid preparation of tissue sections

• Good preservation of antigenicity

• Preservation of fluorescent markers and fluorescent proteins

• Improved retention of lipids and soluble components

• Compatibility with a wide range of histological and molecular labelling procedures

Sample Preparation

Sample preparation must be adapted to the type of tissue and the experimental objective. Samples may be sectioned fresh-frozen or after fixation and cryoprotection.

Rapid and homogeneous freezing is essential to minimize the formation of ice crystals and preserve tissue morphology. Depending on the specimen, sucrose infiltration or another cryoprotective treatment may be recommended before freezing.

The optimal cutting temperature and section thickness depend on tissue composition. Fat-rich, fibrous, calcified, highly hydrated or heterogeneous samples may require specific optimization.

Factors Affecting Section Quality

The quality of cryostat sections may be influenced by:

• Freezing speed and method

• Formation of ice crystals

• Chamber and specimen temperature

• Tissue fixation and cryoprotection

• Blade condition and cutting angle

• Section thickness

• Cutting speed

• Tissue consistency

• Sample orientation

• Slide adhesion

Limitations

Frozen sections generally provide lower morphological detail than well-prepared paraffin- or resin-embedded sections. In addition, poorly controlled freezing may cause ice-crystal artefacts and tissue distortion.

Difficult, very hard or highly heterogeneous specimens may require preliminary testing and optimization.

Epoxy resin embedding is a standard sample-preparation technique used to preserve biological and material specimens for semithin and ultrathin sectioning. It provides excellent mechanical stability and structural preservation, making it particularly suitable for transmission electron microscopy (TEM).

Before embedding, samples are usually chemically fixed, post-fixed, dehydrated through a graded series of ethanol or acetone, and progressively infiltrated with liquid epoxy resin. The resin is then polymerized to produce a hard and stable block that can be trimmed and sectioned using a microtome or ultramicrotome.

Applications

Epoxy resin embedding is suitable for (among others):

• Plant, animal and microbial tissues

• Cell cultures and biological aggregates

• Bacteria, fungi and other microorganisms

• Biomaterials and composite materials

• Nanoparticle-containing samples

• Semithin sectioning for light microscopy

• Ultrathin sectioning for TEM

• Serial sectioning and ultrastructural studies

• Correlative light and electron microscopy

Main Advantages

Epoxy resins provide:

• Excellent preservation of cellular and subcellular ultrastructure

• High mechanical stability during sectioning

• Good support for fragile or heterogeneous samples

• Production of high-quality semithin and ultrathin sections

• Compatibility with conventional heavy-metal contrast staining

• Long-term preservation of embedded specimens

General Procedure

The embedding process generally includes:

• Primary chemical fixation

• Post-fixation, commonly with osmium tetroxide

• Dehydration with graded organic solvents

• Resin infiltration

• Sample orientation in embedding moulds

• Resin polymerization

• Block trimming and sectioning

The exact protocol is adapted to the sample type, size, composition and scientific objective.

Factors Affecting Embedding Quality

The quality of epoxy resin embedding may be influenced by:

• Sample size and permeability

• Fixation and post-fixation conditions

• Dehydration rate

• Resin viscosity

• Infiltration time

• Sample orientation

• Polymerization temperature and duration

• Presence of residual water or solvent

• Hardness of the final resin block

Incomplete infiltration may result in soft areas, cracks, poor sectioning or loss of structural detail.

Limitations

Epoxy resin embedding requires extensive sample processing and is not compatible with live-cell observation. Dehydration and polymerization may alter or extract some soluble components.

In addition, conventional epoxy resins may reduce antigen accessibility, making them less suitable for certain immunolabelling applications. In such cases, alternative acrylic resins or modified fixation protocols may be considered.

LR Gold is a low-viscosity acrylic resin designed for the low-temperature embedding of biological samples.

It is particularly suitable for studies in which the preservation of antigenicity, enzyme activity or other temperature-sensitive cellular components is essential.

Unlike conventional epoxy resins, LR Gold can be infiltrated and polymerized at low temperatures.

Polymerization may be performed using visible light and a light-sensitive initiator, such as benzil, at temperatures as low as approximately −25 °C. These conditions help reduce the loss or alteration of fixation-sensitive and temperature-sensitive antigens.

Applications

LR Gold embedding is particularly suitable for:

• Immunocytochemistry and immunohistochemistry

• Post-embedding immunogold labelling

• Enzyme histochemistry

• Localization of fixation-sensitive antigens

• Preservation of temperature-sensitive enzymes and epitopes

• Light microscopy of resin sections

• Transmission electron microscopy

• Correlative light and electron microscopy

• Analysis of plant, animal and microbial samples

• Studies requiring minimal chemical fixation

The resin can be used for both unfixed and lightly fixed samples, depending on the experimental objective and the degree of structural preservation required.

Sample Preparation

The general preparation procedure may include:

• Collection and trimming of the biological specimen

• No fixation or mild chemical fixation, depending on the application

• Cryoprotection or osmotic protection when required

• Low-temperature dehydration

• Progressive infiltration with LR Gold resin

• Orientation of the sample in an embedding mould or capsule

• Low-temperature light polymerization

• Semithin or ultrathin sectioning

For unfixed tissues, polyvinylpyrrolidone may be used during processing to reduce osmotic changes and help preserve tissue organization.

The exact protocol must be adapted to the sample type, target molecule, antibody, imaging method and required level of morphological preservation.

Sectioning

Polymerized LR Gold blocks can be sectioned using a microtome or ultramicrotome.

Semithin resin sections, commonly approximately 1–4 µm thick, may be collected on glass slides for light microscopy, enzyme histochemistry or immunolabelling.

Ultrathin sections can be collected on electron-microscopy grids for ultrastructural examination and post-embedding immunogold labelling.

Low-temperature LR Gold embedding has been successfully used for the immunogold localization of antigens in ultrathin sections.

Main Advantages

LR Gold provides several advantages:

• Low-temperature resin infiltration and polymerization

• Good preservation of antigenicity

• Improved preservation of temperature-sensitive enzymes and epitopes

• Possibility of embedding unfixed or lightly fixed tissues

• Low viscosity and effective penetration into biological samples

• Compatibility with immunocytochemical techniques

• Compatibility with post-embedding immunogold labelling

• Production of semithin and ultrathin sections

• Suitability for both light and electron microscopy

Limitations

LR Gold embedding must be carefully optimized because the reduction or omission of chemical fixation may compromise ultrastructural preservation.

Other potential limitations include:

• Lower structural preservation than conventional epoxy embedding in some samples

• Sensitivity of the polymerization process to temperature and light conditions

• Risk of incomplete polymerization

• Possible changes in tissue morphology during dehydration and infiltration

• Requirement for specific low-temperature processing equipment

• Need to optimize fixation and labelling conditions for each antibody

• Potential incompatibility with some stains, reagents or analytical procedures

The balance between antigen preservation and ultrastructural preservation should be evaluated according to the scientific objective.

Factors Affecting Embedding Quality

The quality of LR Gold embedding may depend on:

• Sample size and permeability

• Type and duration of fixation

• Dehydration conditions

• Processing temperature

• Resin infiltration time

• Concentration and type of polymerization initiator

• Intensity and duration of light exposure

• Exclusion of oxygen during polymerization

• Sample orientation

• Final hardness of the polymerized block

Incomplete infiltration or polymerization may produce soft blocks, section compression, tearing or loss of morphological detail.

Immunolocalization techniques use antibodies to detect and determine the spatial distribution of specific proteins, antigens or other molecular targets within cells and tissues. Depending on the required resolution, immunolabelling can be examined by light microscopy, fluorescence or confocal microscopy, or transmission electron microscopy.

These approaches combine molecular specificity with morphological information, allowing target molecules to be localized at tissue, cellular or subcellular levels.

Immunolocalization by Light Microscopy

In light-microscopy immunolocalization, a primary antibody specifically recognizes the target antigen. Detection is then performed using a labelled primary antibody or, more commonly, a labelled secondary antibody directed against the primary antibody.

Detection Methods

The main detection approaches include:

• Immunofluorescence, using antibodies conjugated to fluorophores

• Chromogenic immunohistochemistry, using enzyme-conjugated antibodies and substrates that produce a coloured precipitate

• Immunocytochemistry, for detecting targets in isolated cells or cell cultures

• Single and multiple immunolabelling, allowing one or several molecular targets to be examined

• Confocal immunofluorescence, providing optical sectioning and improved spatial localization within thicker samples

Immunofluorescence can be used to determine the cellular or subcellular distribution and relative abundance of specific proteins.

Experimental conditions must be optimized to distinguish specific labelling from background fluorescence and non-specific antibody binding.

Types of Samples

Light-microscopy immunolocalization can be applied to:

• Paraffin-embedded tissue sections

• Cryostat sections

• Resin semithin sections, depending on resin compatibility

• Cultured cells

• Cell aggregates and organoids

• Plant, animal and microbial samples

• Whole-mount preparations

• Free-floating tissue sections

Applications

Applications include (among others):

• Localization of proteins and other antigens

• Identification of specific cell types

• Study of protein expression and distribution

• Analysis of cellular differentiation

• Detection of structural and molecular changes caused by experimental treatments

• Study of host–pathogen interactions

• Colocalization of different molecular targets

• Evaluation of intracellular trafficking

• Selection of regions of interest for subsequent electron microscopy

• Quantitative analysis of fluorescence or chromogenic labelling

Immunolocalization by Electron Microscopy

Immunoelectron microscopy enables the localization of specific antigens at the ultrastructural level.

The most common approach is immunogold labelling, in which antibodies are associated with electron-dense colloidal gold particles that can be directly visualized by TEM.

The technique allows target molecules to be localized in relation to membranes, organelles, vesicles, cell walls and other subcellular structures.

Immunogold Labelling

Immunogold detection may be performed using:

• Primary antibodies directly conjugated to gold particles

• Unlabelled primary antibodies detected with gold-conjugated secondary antibodies

• Gold particles of different diameters for multiple labelling

• Silver enhancement of very small gold particles, when required

Indirect immunogold labelling generally provides stronger signal amplification, whereas direct labelling can reduce the distance between the antigen and the visible gold particle and therefore improve localization precision.

Pre-Embedding Immunolabelling

In pre-embedding immunolabelling, antibodies are applied before resin embedding and ultrathin sectioning.

This approach can provide high labelling sensitivity because antigens are exposed before embedding. However, antibody penetration through thicker samples may be limited, and permeabilization conditions must be carefully controlled to preserve cellular ultrastructure.

Post-Embedding Immunolabelling

In post-embedding immunolabelling, the sample is first embedded and sectioned.

Antibodies are then applied to the surface of ultrathin sections collected on electron-microscopy grids.

This method allows different sections from the same embedded sample to be labelled with different antibodies.

Acrylic resins such as LR Gold are frequently selected because they generally provide better antigen preservation and accessibility than conventional epoxy resins.

Applications of Immuno-TEM

Immunogold electron microscopy can be used for:

• Ultrastructural localization of proteins and antigens

• Identification of proteins associated with specific organelles

• Localization of membrane and cell-wall components

• Analysis of vesicular transport and secretion

• Study of host–pathogen interactions

• Detection of viral or microbial antigens

• Localization of proteins in plant, animal and microbial cells

• Analysis of nanoparticles or labelled biomaterials

• Multiple antigen localization using different gold-particle sizes

• Quantification of immunogold-labelling density and distribution

General Workflow

An immunolocalization study generally includes:

• Selection and validation of primary antibodies

• Optimization of sample collection and fixation

• Selection of the embedding or sectioning method

• Blocking of non-specific binding sites

• Incubation with primary antibodies

• Incubation with fluorescent, enzymatic or gold-conjugated detection reagents

• Washing and counterstaining or contrasting

• Image acquisition

• Evaluation of positive and negative controls

• Qualitative or quantitative image analysis

The exact procedure must be adapted to the target antigen, antibody, tissue type, fixation method, embedding medium and imaging technique.

Controls

Appropriate controls are essential for evaluating antibody specificity and distinguishing true antigen localization from non-specific labelling.

Recommended controls may include:

• Omission of the primary antibody

• Replacement of the primary antibody with an appropriate non-immune immunoglobulin

• Positive control samples known to contain the antigen

• Negative control samples lacking the antigen

• Antibody pre-adsorption controls, when suitable

• Single-labelling controls for multiple immunofluorescence experiments

• Evaluation of endogenous fluorescence or enzymatic activity

• Comparison of labelling over the target structure and surrounding background

Antibody validation and strict assessment of non-specific labelling are particularly important when interpreting immunolocalization results.

Factors Affecting Labelling Quality

The quality of immunolocalization may depend on:

• Antibody specificity and affinity

• Antibody concentration

• Type and duration of fixation

• Antigen accessibility

• Tissue permeability

• Embedding medium

• Section thickness

• Blocking conditions

• Incubation time and temperature

• Washing conditions

• Autofluorescence

• Non-specific antibody binding

• Preservation of cellular morphology

• Imaging and acquisition settings

Strong fixation may provide excellent structural preservation but reduce antigen accessibility. Conversely, mild fixation may improve antibody recognition while compromising tissue or ultrastructural preservation. The protocol must therefore balance antigenicity and morphology.

Advantages

Immunolocalization provides:

• Specific detection of molecular targets

• Localization within tissues, cells and organelles

• Combination of molecular and morphological information

• Possibility of single or multiple labelling

• Compatibility with qualitative and quantitative image analysis

• High spatial resolution when immunogold TEM is used

• Integration with fluorescence, confocal and electron microscopy

• Potential application in correlative light and electron microscopy

Limitations

Potential limitations include:

• Dependence on antibody specificity and quality

• Loss or masking of epitopes during fixation and embedding

• Limited antibody penetration in thick samples

• Autofluorescence and non-specific background

• Reduced ultrastructural preservation under mild fixation conditions

• Lower labelling efficiency in some resin-embedded samples

• Limited accessibility of antigens located inside the resin section

• Requirement for extensive optimization and appropriate controls

• Risk of interpreting gold particles located close to, but not directly over, the target structure

Immunolabelling results should be interpreted together with sample morphology, controls and background-labelling levels.