The quality of histological samples depends not only on the staining step but also on upstream and downstream procedures including fixation, dehydration, clearing, embedding, deparaffinization, and mounting. Different reagent systems vary markedly in tissue morphology preservation, antigen retention, medium compatibility, and microscopic imaging quality. Therefore, a continuous and compatible processing workflow should be established according to sample type and downstream detection objectives.

Keywords: tissue fixation; tissue clearing; deparaffinization; paraffin section; H&E staining; mounting; microscopic slide preparation

1 Technical Workflow of Histological Sample Preparation

1.1 Tissue Fixation

After tissue removal, autolysis, protein degradation, and structural disruption can occur. Fixation limits these changes through protein crosslinking or precipitation, preserving cellular and tissue structures as close as possible to their state at the time of collection. The fixation method needs to be compatible with subsequent paraffin embedding, frozen sectioning, H&E staining, special staining, IHC, or IF.

1.2 Dehydration and Clearing

Fixed tissues contain large amounts of water, whereas paraffin and most resin-based mounting media are incompatible with water. Therefore, paraffin tissue processing usually requires graded ethanol dehydration followed by a clearing agent to complete medium conversion between ethanol and paraffin. Inadequate clearing can lead to insufficient paraffin infiltration, reduced section quality, and abnormal staining background.

1.3 Embedding and Sectioning

Paraffin embedding involves infiltrating cleared tissue with molten paraffin to form an embedding block with sufficient mechanical strength for obtaining micrometer-scale serial sections. Embedding quality is affected by the completeness of preceding fixation, dehydration, and clearing steps. Therefore, section fragmentation, voids, or tissue detachment are not necessarily caused by the sectioning equipment itself.

1.4 Deparaffinization and Rehydration

Before paraffin sections enter most aqueous staining systems, paraffin needs to be removed, followed by gradual rehydration from absolute ethanol to lower ethanol concentrations. Residual paraffin creates a hydrophobic barrier that restricts penetration of Hematoxylin, Eosin, and other aqueous stains into tissues and is an important pretreatment factor contributing to uneven staining.

1.5 Staining and Mounting

Staining increases structural contrast through interactions between different dyes and nuclei, cytoplasm, connective tissue, or specific chemical components. After staining, dehydration, clearing, and mounting conditions are selected according to whether an aqueous or resin-based mounting system is used, allowing tissue sections to be preserved long term while maintaining stable microscopic observation quality.

2 Characteristics and Selection of Fixation Systems

2.1 Paraformaldehyde Fixation

(1) Fixation Characteristics

After releasing formaldehyde, Paraformaldehyde forms crosslinks with proteins and can preserve overall cellular and tissue structures relatively well. 4% Paraformaldehyde is a common fixation condition in cell, tissue, and immunostaining studies. Its crosslinking strength is relatively moderate and can balance morphology preservation with the requirements of most downstream assays.

(2) Major Advantages

Paraformaldehyde has good compatibility with tissue morphology and many protein antigens and is suitable for IHC, IF, frozen sections, and routine histology. Compared with strong crosslinking fixatives such as Glutaraldehyde, its effects on antigen epitope accessibility are generally more readily compensated for through antigen retrieval or condition optimization.

(3) Major Limitations

Excessively long fixation increases protein crosslinking, masking some antigen epitopes and reducing nucleic acid and protein extraction efficiency. Formaldehyde fixation has limited direct ability to preserve lipids and therefore cannot meet all requirements for lipid morphology or membrane-component studies.

2.2 Neutral Buffered Formalin

(1) Fixation Characteristics

Neutral Buffered Formalin uses formaldehyde as its major active component and maintains a relatively stable pH through a buffering system. It is a well-established fixation system for routine pathological paraffin sample processing.

(2) Major Advantages

It provides good tissue penetration, morphology preservation, and compatibility with paraffin processing and is suitable for routine pathological tissues, H&E staining, and most IHC workflows. For experiments requiring standardized batch tissue processing, the neutral buffered system provides good reproducibility.

(3) Major Limitations

Prolonged fixation can increase protein crosslinking and affect certain antigen epitopes and nucleic acid quality. Excessively thick tissue blocks may also show sufficient fixation at the periphery but inadequate fixation in the center. Therefore, tissue size, fixative volume, and processing time need to be controlled together.

2.3 Glutaraldehyde Fixation

(1) Fixation Characteristics

Glutaraldehyde has strong protein-crosslinking ability and can form stable molecular networks, providing strong fixation of cell membranes, organelles, and ultrastructure.

(2) Major Advantages

Glutaraldehyde is particularly suitable for ultrastructural studies such as Transmission Electron Microscopy and can preserve details including mitochondria, endoplasmic reticulum, membrane structures, and cell junctions relatively well.

(3) Major Limitations

Strong crosslinking markedly reduces accessibility of some antigens and may generate substantial autofluorescence. Therefore, it is usually not the preferred fixation system for routine IF and most IHC experiments. Its tissue penetration is also relatively slow, and tissue size needs to be carefully controlled, particularly for thicker samples.

2.4 Carnoy and Clarke Alcohol-Acid Fixation Systems

(1) Fixation Characteristics

Carnoy and Clarke systems mainly use alcohols such as ethanol to induce protein precipitation, while components such as acetic acid improve preservation of nuclear structures. Their fixation mechanisms differ from aldehyde-based crosslinking systems.

(2) Major Advantages

These systems provide rapid fixation and relatively good preservation of nuclei, chromatin, and some nucleic acid structures, making them suitable for studies emphasizing nuclear morphology, nucleic acid localization, or specific cytological structures.

(3) Major Limitations

Alcohol treatment causes substantial dehydration and tissue shrinkage and can extract lipids. Therefore, preservation of cytoplasm, membrane systems, and overall tissue morphology is generally weaker than with formaldehyde-based systems. These fixatives should not be prioritized for experiments requiring intact membrane structures or lipid components.

2.5 Ethanol Fixation

Ethanol achieves rapid fixation through dehydration and protein precipitation. It is simple to use and suitable for some cell smears, cytological staining, and nucleic acid-related experiments. Its major disadvantages are marked tissue shrinkage and limited preservation of organelles and membrane structures. Therefore, it should not simply replace formaldehyde for routine morphological evaluation of paraffin-embedded tissues.

Table 1 Characteristics and Selection of Common Fixation Systems

3 Dehydration Systems and Processing Intensity

3.1 Graded Ethanol Dehydration

(1) Functional Characteristics

Graded ethanol gradually replaces water within tissues by progressively increasing the ethanol concentration. Gradients such as 70%, 80%, 90%, 95%, and absolute ethanol reduce abrupt tissue shrinkage and morphological changes that can occur when tissues move directly from an aqueous environment into an anhydrous environment.

(2) Major Advantages

Progressive dehydration helps preserve tissue morphology and improves uniform penetration of subsequent clearing agents, making it a highly stable dehydration method for paraffin embedding workflows. For thicker tissues, extending the duration of intermediate gradients is generally more favorable for uniform dehydration than simply prolonging the absolute ethanol step.

(3) Major Limitations

Graded dehydration involves multiple steps, and prolonged exposure to high concentrations of ethanol can still cause tissue hardening, shrinkage, and increased brittleness. Tissue size, lipid content, degree of fibrosis, and prior fixation conditions all affect the appropriate dehydration time.

3.2 Inadequate Dehydration

Residual water in tissues reduces penetration efficiency of Xylene or environmentally friendly clearing agents, resulting in incomplete tissue clearing, focal whitening, difficulty with paraffin infiltration, or voids in sections. These problems cannot be compensated for simply by extending paraffin infiltration time. The adequacy of high-concentration ethanol dehydration and whether absolute ethanol has absorbed water and lost effectiveness should be checked first.

3.3 Over-Dehydration

Excessive dehydration can make tissues hard, brittle, and markedly shrunken, with particularly pronounced effects on small and soft tissues. The dehydration endpoint should aim to complete water replacement while maintaining structural integrity rather than simply extending exposure to absolute ethanol.

4 Types and Differences of Clearing Systems

4.1 Lactic Acid Clearing Solution

(1) Functional Characteristics

Lactic Acid Clearing Solution is mainly used for some plant disease tissues and whole-mount samples. By reducing tissue background and improving light transmission, it facilitates microscopic observation of hyphae, spores, and other internal structures.

(2) Major Advantages

Samples can retain relatively intact spatial structures without paraffin embedding or sectioning. This makes the method suitable for observing pathogen distribution within leaves or other plant tissues and internal structures of whole tissues.

(3) Major Limitations

Lactic acid clearing is generally relatively slow, and its primary objective is whole-tissue observation. It does not perform ethanol–paraffin medium conversion or paraffin dissolution during paraffin tissue processing and therefore cannot replace conventional paraffin deparaffinization reagents.

4.2 Lactophenol Clearing System

(1) Functional Characteristics

The Lactophenol system combines tissue clearing with observation of fungal structures. When used together with stains such as Lactophenol Blue, it can enhance microscopic contrast of hyphae, spores, and related structures.

(2) Major Advantages

It is suitable for fungal morphology and plant pathogenic fungi, and clearing and staining can form a continuous processing workflow. It is particularly suitable for microorganisms or whole-tissue samples that do not require paraffin embedding.

(3) Major Limitations

Its application is relatively specialized. It is not a general-purpose clearing system for routine paraffin processing of animal tissues and should not replace Xylene or environmentally friendly paraffin clearing agents.

4.3 Xylene Clearing System

(1) Functional Characteristics

Xylene has good compatibility with both absolute ethanol and paraffin and can be used for clearing before paraffin infiltration, deparaffinization of paraffin sections, and clearing after staining before resin mounting.

(2) Major Advantages

It has strong paraffin-dissolving capacity and high medium-conversion efficiency. Traditional paraffin-processing workflows are well established, and Xylene is compatible with most resin-based mounting media.

(3) Major Limitations

Xylene is relatively volatile and irritating and requires good ventilation and appropriate laboratory exposure controls. Excessive exposure of tissues to Xylene can also increase tissue hardening and brittleness.

4.4 Environmentally Friendly Paraffin Infiltration, Deparaffinization, and Clearing Solution

(1) Functional Characteristics

Environmentally friendly paraffin infiltration, deparaffinization, and clearing solutions are used to replace some traditional aromatic clearing agents and can cover multiple stages including clearing before paraffin infiltration, deparaffinization of paraffin sections, and post-staining clearing.

(2) Major Advantages

These reagents provide broad functional coverage and can perform multiple medium-conversion steps in paraffin tissue processing within a single system while reducing the use of traditional aromatic clearing solvents. They are suitable for routine paraffin processing and some IHC workflows.

(3) Major Limitations

Different paraffin formulations, section thicknesses, and tissue-processing procedures affect actual deparaffinization efficiency. After changing clearing agents, deparaffinization time, tissue morphology, and staining consistency still need to be validated rather than mechanically applying the original Xylene processing time.

4.5 Environmentally Friendly Deparaffinization Solution

Environmentally friendly deparaffinization solutions are more specifically focused on paraffin removal and pretreatment before staining and are suitable for sectioning workflows that aim to reduce the use of conventional Xylene. Compared with environmentally friendly paraffin infiltration, deparaffinization, and clearing solutions that can also perform pre-infiltration clearing, their experimental positioning is more focused on the section stage. Therefore, selection should clearly distinguish between the need for a “full-process clearing medium” and a “deparaffinization-only system.”

Table 2 Comparison of Common Clearing and Deparaffinization Systems

5 Paraffin Embedding and Section Quality

5.1 Paraffin Infiltration

Cleared tissues are transferred into molten paraffin, allowing the clearing medium to be gradually replaced by paraffin. Paraffin needs to sufficiently penetrate tissue spaces and form uniform support. Insufficient infiltration can leave the tissue center soft and cause section breakage, whereas excessively high temperature or prolonged processing can increase tissue hardening and affect some antigens.

5.2 Embedding Quality

Embedding orientation, paraffin condition, and completeness of tissue infiltration jointly determine the subsequent cutting plane. Samples such as skin, gastrointestinal tract, tubular tissues, and tissues with distinct layers need to be oriented according to the observation objective. Otherwise, even an intact section may fail to provide tissue structures with analytical value.

5.3 Sources of Sectioning Abnormalities

Fragmentation of serial sections, voids, tissue detachment, or uneven section surfaces are not necessarily caused by the microtome or blade. Overfixation, insufficient dehydration, excessive tissue hardness, inadequate paraffin infiltration, and incorrect embedding orientation can all produce similar problems and need to be traced through the preprocessing workflow.

6 Deparaffinization and Rehydration of Paraffin Sections

6.1 Completeness of Deparaffinization

Paraffin in sections prevents aqueous stains from entering tissues, so deparaffinization must be completed before rehydration. Incomplete deparaffinization commonly manifests as localized liquid repellence, weak H&E staining, patchy staining, or uneven IHC reactions and is a frequent pretreatment abnormality in paraffin staining.

6.2 Rehydration Gradient

After removal of the clearing agent, sections need to pass through absolute ethanol, high-concentration ethanol, and lower-concentration ethanol before entering the aqueous phase, allowing tissues to gradually readapt to the aqueous staining environment. Stable gradient medium conversion reduces morphological changes and improves uniform penetration of aqueous stains.

6.3 Selection of Deparaffinization Reagents

Xylene is suitable for established traditional paraffin workflows, with rapid deparaffinization and good compatibility with resin mounting. Environmentally friendly deparaffinization reagents are more suitable for laboratories aiming to reduce aromatic solvent use. In addition to deparaffinization speed, selection should compare tissue morphology, staining background, antigen preservation, and compatibility with existing automated staining workflows.

7 Hematoxylin and Eosin Staining Systems

7.1 Hematoxylin Staining Systems

(1) Mayer Hematoxylin

The Mayer system is generally a relatively mild progressive nuclear staining system with low background and is suitable for routine nuclear staining and IHC counterstaining. Its advantage is that nuclear staining intensity is relatively easy to control and differentiation requirements are relatively low. Its limitation is that deeper nuclear staining generally requires longer processing times.

(2) Harris Hematoxylin

The Harris system provides rapid and strong nuclear staining and is suitable for routine H&E and samples requiring prominent nuclear contrast. Its major limitation is a tendency toward overstaining, requiring strict control of staining and differentiation times.

(3) Gill Hematoxylin

Gill formulations provide different staining intensities depending on concentration and are suitable for histological and cytological nuclear staining. Their advantage is a relatively high degree of standardization, with different formulations available according to sample and application. However, staining time still requires optimization across tissues of different thicknesses and different staining procedures.

(4) Cole Hematoxylin

The Cole system can be used for routine histological nuclear staining. Its suitability should be evaluated according to nuclear staining intensity, background, differentiation requirements, and overall color contrast with Eosin rather than directly replacing other Hematoxylin systems using the same staining time.

7.2 Eosin Staining Systems

(1) Water-Soluble Eosin

Water-soluble Eosin binds cytoplasm and multiple protein structures under aqueous conditions, is easy to use, and is suitable for routine H&E staining. After staining, ethanol dehydration is still required, and the duration of subsequent ethanol treatment continues to affect final Eosin intensity.

(2) Alcohol-Soluble Eosin

Alcohol-soluble Eosin can be integrated more directly with ethanol dehydration systems and is suitable for some paraffin H&E procedures. Its advantage is relatively simple medium conversion, but staining performance is sensitive to ethanol concentration, staining-solution condition, and treatment time.

Table 3 Selection Characteristics of Common H&E Staining Systems

8 Characteristics and Selection of Mounting Systems

8.1 Canada Balsam

Canada Balsam is a traditional resin-based permanent mounting medium with good optical transparency and is suitable for tissue sections that have completed dehydration and clearing. Its advantages are stable slide preparation and suitability for long-term bright-field preservation. Its limitation is that samples must be completely dehydrated and cleared and it is not suitable for direct use with aqueous samples.

8.2 Neutral Balsam

Neutral Balsam is widely used for permanent mounting after H&E and other routine tissue staining and integrates well with Xylene-based clearing systems. It has an established workflow and stable imaging performance, but residual water or low-concentration ethanol in sections can readily cause whitening, turbidity, and refractive abnormalities.

8.3 Glycerol-Based Systems

Glycerol-based media are suitable for some aqueous temporary preparations and whole-mount observation of plant and microbial samples and can eliminate complicated dehydration and organic clearing procedures. Their advantages include simple operation and compatibility with aqueous samples, but long-term storage stability and mechanical stability are generally lower than those of resin-based permanent mounting systems.

8.4 Antifade Mounting Medium

Antifade mounting systems are used for IF and other fluorescent samples, with the primary objective of delaying Fluorophore photobleaching and preserving fluorescence signals rather than increasing refractive transparency in conventional bright-field sections. Their selection should match the fluorescent dye, microscopy method, and storage duration and they cannot simply replace resin-based mounting media used for H&E.

Table 4 Comparison of Common Mounting Systems

9 Common Slide-Preparation Abnormalities and Result Interpretation

9.1 Insufficient Clearing or Paraffin Infiltration in the Tissue Center

Whitening in the tissue center, voids in sections, or localized loose structures generally indicate insufficient preceding dehydration and clearing. Priority should be given to checking tissue-block thickness, adequacy of high-concentration ethanol dehydration, whether clearing time is appropriate, and whether the clearing agent has absorbed water or lost effectiveness.

9.2 Localized Liquid Repellence or Weak Staining

Incomplete deparaffinization causes residual paraffin to block aqueous stain penetration, producing localized water repellence, weak staining, or patchy staining. The type of deparaffinization reagent, treatment time, temperature, and degree of reagent reuse should be compared, and the workflow should be adjusted if paraffin formulation or section thickness has changed.

9.3 Excessively Hard or Fragmented Tissue

Excessive tissue hardness can result from overfixation, over-dehydration, prolonged clearing, or excessive paraffin infiltration temperature. Multiple steps can produce similar phenotypes, so the entire preprocessing workflow needs to be traced according to the stage at which the problem appears rather than relying only on blade replacement or section-thickness adjustment.

9.4 Excessively Dark or Light Nuclear Staining

Excessively dark nuclear staining is generally associated with prolonged Hematoxylin exposure or insufficient differentiation, whereas weak nuclear staining may result from reduced stain activity, incomplete deparaffinization and rehydration, or excessive differentiation. Interpretation requires simultaneous consideration of stain condition and pretreatment completeness.

9.5 Turbidity After Mounting

When whitening, uneven refraction, or localized turbidity occurs after resin mounting, residual water or low-concentration ethanol, completeness of post-staining dehydration, degree of clearing, and compatibility between the mounting medium and clearing agent should be checked. Simply increasing the amount of mounting medium generally cannot resolve incomplete medium conversion.

10 Products Related to Tissue Fixation, Clearing, Deparaffinization, Staining, and Mounting

10.1 Fixation and Tissue Pretreatment Products

10.2 Clearing, Deparaffinization, and Medium Conversion Products

10.3 Staining and Microscopic Observation Products

10.4 Mounting-Related Products

There is no single histological processing system suitable for all samples. The fixation stage requires balancing tissue morphology, antigen accessibility, and ultrastructure preservation; the clearing and deparaffinization stages require consideration of medium-conversion efficiency, paraffin-dissolving capacity, and the risk of tissue hardening; and staining and mounting should be selected according to bright-field, IHC, or fluorescence imaging objectives.

For more related articles, please see below:

[1] Histological and Cytological Section Staining: Principles and Methodological Essentials for Routine Pathology Stains and Structural Special Stains

[2] Analysis of Classical Histological Special Staining Systems: Application Selection of PTAH, Masson, VG, Elastic Fiber Staining, and Reticular Fiber Staining

[3] How to Choose Common Fixatives? A Comparison of Fixation Strategies for Histopathology, Immunostaining, Cytology, and Electron Microscopy Samples

[4] Application Differences Among Glutaraldehyde, Paraformaldehyde, and Formalin in Tissue and Cell Sample Fixation

[5] Cell fixation sectioning and embedding experiments

[6] Hematoxylin–Eosin (H&E) Staining Standard Operating Procedure (SOP)