Histological Table

Method	Application	Advantage	Disadvantage	Observation	Image
Cryostat(s) (7µm to 40µm) Sections of fixed or unfixed tissues Freezing with liquid nitrogen	Fluorescence or enzymatic immunostaining In situ hybridization Staining	Rapid technique, ideal for detecting “difficult” antigens, liposoluble antigens… Can be used on fixed or unfixed tissues No degradation of antigenic sites: Protein (enzyme, receptor, mRNA …) chemical substance (neurotransmitter…) Ø No embedding Ø No dehydration Ø No polymerization Ø No heat Ø No chemical reactions	Technique is difficult to master, sections often irregular Morphological preservation varies depending on tissue, presence of ice crystals (holes)	Light microscopy Fluorescence microscopy (ApoTome)	Rat Corti organ section
Microtome(s) (4µm to 20µm) Sections of fixed tissues Paraffin embedding	– Histological staining Pathological study Qualitative and quantitative study Serial sections: image analysis and counting Immunohistochemistry	Excellent morphology Technique of choice for all histological staining	Immunohistochemistry: 1- Heat-sensitive antigens 2- Sensitive to dehydration 3- Sensitive to chemical reactions (lipid precipitation…) 4- Sensitive to paraffin embedding and polymerization (antigen retrieval required) Heavy embedding, automated embedding recommended Immunofluorescence not suitable (background noise)	Light microscopy Slide scanner – Nanozoomer	Mouse knee section
Ultramicrotome(s) (0.25µm to 2µm) Sections of fixed tissues Epoxy resin embedding (50nm to 100nm)	Light microscopy, 1µm thin section: 1- Structure contrast with toluidine blue 2- Qualitative and quantitative study, serial sections: image analysis and counting Transmission electron microscopy (TEM): 1- Immunostaining of fine structures in TEM: “Pre-embedding and Post-embedding”	Only histological technique allowing ultrastructural observation Excellent morphology For immunostaining: tissue-specific optimization required	Time-consuming technique Strict preparation parameters required Requires experienced technician	Light microscopy TEM Slide scanner – Nanozoomer	Ultrastructural immunocytochemistry in pre-embedding (performed before epoxy resin embedding)
Vibratome(s) (50µm to 500µm) Thick sections of fixed or living tissues Gel embedding possible for small samples	Fluorescence immunostaining In situ hybridization Electrophysiology (patch-clamp…) Culture: explants Preparation of sections for TEM embedding	Very thick sections (50µm to 500µm) Sections on living tissues (cooling bath at 4°C) or fixed tissues Excellent morphology Good immunofluorescence 3D reconstruction possible	Difficult technique to master: tissue-specific optimization needed Challenges with cutting heterogeneous hardness samples Issues with antibody penetration in thick sections Antigen retrieval required	3D Confocal Microscopy Two-photon Microscopy	Rat vestibular crest section (photo by Aurore Brugeaud)

Method

Application

Advantage

Disadvantage

Observation

Image

Cryostat(s)

(7µm to 40µm)
Sections of fixed or unfixed tissues

Freezing with liquid nitrogen

Fluorescence or enzymatic immunostaining
In situ hybridization
Staining

Rapid technique, ideal for detecting “difficult” antigens, liposoluble antigens…

Can be used on fixed or unfixed tissues

No degradation of antigenic sites: Protein (enzyme, receptor, mRNA …) chemical substance (neurotransmitter…)

Ø No embedding
Ø No dehydration
Ø No polymerization
Ø No heat
Ø No chemical reactions

Technique is difficult to master, sections often irregular

Morphological preservation varies depending on tissue, presence of ice crystals (holes)

Light microscopy

Fluorescence microscopy (ApoTome)

Rat Corti organ section

Microtome(s)

(4µm to 20µm)
Sections of fixed tissues

Paraffin embedding

– Histological staining
Pathological study
Qualitative and quantitative study
Serial sections: image analysis and counting
Immunohistochemistry

Excellent morphology

Technique of choice for all histological staining

Immunohistochemistry:

1- Heat-sensitive antigens
2- Sensitive to dehydration
3- Sensitive to chemical reactions (lipid precipitation…)
4- Sensitive to paraffin embedding and polymerization (antigen retrieval required)
Heavy embedding, automated embedding recommended
Immunofluorescence not suitable (background noise)

Light microscopy
Slide scanner – Nanozoomer

Mouse knee section

Ultramicrotome(s)

(0.25µm to 2µm)
Sections of fixed tissues

Epoxy resin embedding (50nm to 100nm)

Light microscopy, 1µm thin section:
1- Structure contrast with toluidine blue
2- Qualitative and quantitative study, serial sections: image analysis and counting
Transmission electron microscopy (TEM):
1- Immunostaining of fine structures in TEM: “Pre-embedding and Post-embedding”

Only histological technique allowing ultrastructural observation

Excellent morphology

For immunostaining: tissue-specific optimization required

Time-consuming technique

Strict preparation parameters required

Requires experienced technician

Light microscopy
TEM
Slide scanner – Nanozoomer

Ultrastructural immunocytochemistry in pre-embedding (performed before epoxy resin embedding)

Vibratome(s)

(50µm to 500µm)
Thick sections of fixed or living tissues

Gel embedding possible for small samples

Fluorescence immunostaining

In situ hybridization

Electrophysiology (patch-clamp…)

Culture: explants

Preparation of sections for TEM embedding

Very thick sections (50µm to 500µm)

Sections on living tissues (cooling bath at 4°C) or fixed tissues

Excellent morphology

Good immunofluorescence

3D reconstruction possible

Difficult technique to master: tissue-specific optimization needed

Challenges with cutting heterogeneous hardness samples

Issues with antibody penetration in thick sections

Antigen retrieval required

3D Confocal Microscopy

Two-photon Microscopy

Rat vestibular crest section (photo by Aurore Brugeaud)