A Review of Lipid Histochemistry

Department of Human Anatomy and Cell Biology, Faculty of Basic Medical Sciences, College of Health Sciences, Delta State University, Abraka, Nigeria

*Corresponding Author: Ishokare Philip Oghenerunor, Department of Human Anatomy and Cell Biology, Faculty of Basic Medical Sciences, College of Health Sciences, Delta State University, Abraka, Nigeria, E-mail: [email protected]

Received Date: November 17, 2025

Published Date: December 19, 2025

Citation: Oghenerunor IP, et al. (2025). A Review of Lipid Histochemistry. Mathews J Case Rep. 10(5):215.

Copyrights: Oghenerunor IP, et al. © (2025).

ABSTRACT

Introduction: Lipid histochemistry is a specialized branch of histology that focuses on the identification, localization, and characterization of lipids within biological tissues. Lipids, which include neutral fats, phospholipids, glycolipids, and sterols, play essential roles in cellular structure, energy metabolism, and signaling pathways. Aim: The general objective is to critically examine and synthesize existing literature on lipid histochemistry with the aim of evaluating traditional methods, recent advances, and future perspectives in the visualization and characterization of lipids within biological tissues. Search Strategy: This review followed a comprehensive literature search which was conducted across four electronic databases: PubMed, Scopus, Web of Science, and Google Scholar. The search covered studies published from January 1, 2018, to May 2025. Search terms included various combinations and Boolean operators of keywords such as: "lipid histochemistry", "histological staining of lipids", "lipid visualization techniques", "lipid droplet detection", "Sudan stain", "Oil Red O", "Nile Red", "immunohistochemistry and lipids", "CARS microscopy", and "fluorescent lipid staining". Filters were applied to limit the results to peer-reviewed journal articles in English that provided original experimental data or review-level analyses on histochemical methods for lipid detection. Results: From the systematic search spanning 2018 to 2025, the analysis yielded a broad but thematically coherent body of studies that collectively deepened our understanding of lipid histochemistry. What emerged most clearly was the progressive refinement of techniques that not only detect lipids but also distinguish between their diverse classes neutral fats, phospholipids, glycolipids, and complex derivatives within intact tissues. Conclusion: Despite these advances, several challenges remain. Standardization across laboratories, particularly for novel fluorescence-based or AI-assisted methods, is still lacking. Differences in tissue preparation protocols, staining duration, lipid solubility during fixation, and data interpretation methodologies continue to introduce variability that can hinder reproducibility and clinical translation.

Keywords: Histochemistry, Lipids, Stains, Lipid Localization, Diagnosis, Medical.

INTRODUCTION

Lipids are structurally and functionally indispensable to cellular life [1]. They serve not only as major constituents of biological membranes but also as energy reservoirs and signaling molecules critical to diverse physiological processes [2]. In histological contexts, lipids are increasingly being recognized as dynamic biomarkers of both normal tissue architecture and pathological transformation, necessitating refined techniques for their accurate detection and localization [3].

The field of lipid histochemistry which encompasses the detection, visualization, and spatial mapping of lipid molecules in situ has evolved significantly in recent years, integrating classical dye-based techniques with advanced molecular and imaging methodologies [4]. One of the major challenges in lipid histochemistry lies in the physicochemical nature of lipids themselves. Their hydrophobicity and susceptibility to extraction during routine tissue processing make them difficult to preserve and stain reliably in paraffin-embedded sections.

As a result, frozen sectioning techniques have remained the gold standard for traditional lipid visualization using lipophilic dyes such as Sudan III, Sudan IV, Oil Red O, and Nile Blue [2]. These dyes exhibit strong affinity for triglycerides and cholesterol esters, enabling basic differentiation of neutral lipids. Osmium tetroxide, a heavy metal fixative, is also widely used for its ability to fix and stain unsaturated lipids black, particularly in electron microscopy [5]. However, such conventional methods lack specificity and are unable to resolve different classes of lipids such as phospholipids, glycolipids, or cholesterol-rich domains.

Recent advances have pushed the boundaries of lipid histochemistry beyond traditional staining. Fluorescence-based techniques using dyes like Nile Red has significantly improved sensitivity and specificity, especially for neutral lipids and lipid droplets [6,7]. Raman-based imaging modalitiesincluding Coherent Anti-Stokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS)enable label-free, high-resolution mapping of lipid distribution, offering a non-invasive and quantitative assessment of lipid biochemistry in tissues. Furthermore, immunohistochemistry targeting lipid-metabolizing enzymes, lipid transporters (e.g., CD36, FATPs), and apolipoproteins has emerged as a complementary approach, especially in metabolic and neurodegenerative research [8].

In pathological settings, lipid histochemistry plays an increasingly vital role in diagnosing and understanding disease progression. In cardiovascular pathology, atherosclerotic plaques characterized by foam cells and cholesterol deposits are commonly evaluated using lipid-specific staining [9]. In hepatology, steatosis and non-alcoholic fatty liver disease (NAFLD) are diagnosed through the accumulation of lipid droplets in hepatocytes, visualized by Oil Red O and Nile Red staining [10]. In oncology, lipid droplets have gained attention as metabolic organelles supporting cancer cell survival, chemoresistance, and proliferation, necessitating their histological detection in tumor biopsies [11]. Moreover, lipid dysregulation in the brain has been linked to neurodegenerative diseases, with altered myelin lipid composition evident in multiple sclerosis and Alzheimer's disease [12,13].

As biomedical sciences lean toward molecular precision, lipid histochemistry is now being combined with proteomics, lipidomics, and high-resolution imaging to create multi-dimensional datasets, facilitating better understanding of tissue lipid architecture [14]. However, despite these advances, a unified understanding of the current landscape of lipid histochemical techniques remains limited. Previous reviews have often focused on isolated techniques or specific diseases. Hence, a comprehensive and systematic synthesis of lipid histochemistry literature between 2018 and 2025 is imperative to capture the breadth and evolution of this field. This review aims to consolidate recent methodological advances, highlight their applications across various organ systems and diseases, and identify gaps that future research should address.

Statement of the Problem

Lipids play central roles in energy storage, membrane dynamics, cell signaling, and disease pathology. Despite their importance, studying lipids at the tissue level remains a major challenge in histochemistry. Conventional staining methods such as Sudan dyes and Oil Red O are still widely used, but they offer limited specificity, poor reproducibility, and are unable to differentiate between lipid subclasses with accuracy. On the other hand, modern approaches such as mass spectrometry imaging and vibrational spectroscopy provide higher sensitivity and spatial resolution, yet they are expensive, technically demanding, and inaccessible to many laboratories in resource-limited settings.

This methodological divide has created a persistent gap in lipid histochemistry: most studies remain descriptive, lacking molecular precision, while cutting-edge technologies are underutilized in clinical and research practice. Furthermore, the integration of classical histochemical stains with newer biochemical and imaging techniques is still poorly explored, leaving uncertainties about standardization, comparability, and diagnostic utility. Without addressing these limitations, the full potential of lipid histochemistry in unraveling metabolic disorders, neurodegenerative diseases, and cancer biology remains underdeveloped.

General Objective

The general objective is to critically examine existing literatures on lipid histochemistry by evaluating traditional methods, and recent advances in the visualization and characterization of lipids within biological tissues.

LITERATURE REVIEW

Histochemical methods for lipid detection have significantly evolved over the past decade, responding to a growing demand for accuracy in visualizing and differentiating lipid types within diverse biological tissues. Classical lipid stains such as Sudan Black B, Oil Red O (ORO), and osmium tetroxide have long been employed to identify neutral fats and phospholipids in frozen sections, especially in liver and adipose tissue histology. These methods, despite their limitations in specificity and permanence, remain foundational in many laboratories due to their ease of use and affordability [15,16].

Among classical stains, ORO has retained popularity, particularly in metabolic and hepatic pathology studies. The stain binds predominantly to neutral triglycerides and cholesterol esters, producing a vivid red coloration under light microscopy. In a recent comparative study by [2], ORO staining in murine models of non-alcoholic fatty liver disease (NAFLD) was shown to reliably correspond with biochemical quantifications of hepatic lipid load, reinforcing its diagnostic value. However, its inability to differentiate specific lipid classes or visualize lipid-protein interactions remains a drawback [17].

Sudan Black B, with broader lipid affinity, has also found renewed application in the context of neurohistology, where it is used to identify myelin degeneration and lipofuscin accumulation in neurodegenerative conditions [18]. In Alzheimer's disease models, Sudan Black B staining has been used to visualize lipid peroxidation products within amyloid plaques, offering histological insights into oxidative damage pathways [19]. Osmium tetroxide, traditionally a fixative and lipid stain, remains relevant in electron microscopy, enabling high-resolution visualization of lipid membranes and droplets [20].

In recent years, fluorescence-based histochemistry has gained momentum, particularly with the use of Nile Red and BODIPY (boron-dipyrromethene) dyes. Nile Red is a lipophilic stain that fluoresces in lipid environments, allowing for dynamic quantification of intracellular lipid droplets in both live and fixed cells. Its spectral properties vary with lipid polarity, enabling discrimination between neutral lipids and phospholipidsa feature leveraged in studies of adipogenesis, steatosis, and tumor lipid metabolism [21]. BODIPY-based probes have similarly gained recognition for their stability, photostaining precision, and compatibility with confocal microscopy.

In parallel, immunohistochemical (IHC) approaches have been developed to detect lipid metabolism-related enzymes such as adipophilin (PLIN2), FABP4, ACAT1, and CD36. These proteins serve as proxies for intracellular lipid content and trafficking. Immunostaining for adipophilin, in particular, has been widely used to identify lipid-laden cells in sebaceous glands, adrenal cortex, and breast carcinoma tissues [22]. IHC has enabled pathologists to move beyond lipid accumulation toward understanding the metabolic behavior of cells within tissues.

Additionally, nonlinear optical imaging techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy have revolutionized lipid histochemistry. These label-free modalities utilize vibrational signals of CH bonds to visualize lipid structures in living tissues with subcellular resolution [23]. Studies applying SRS microscopy in atherosclerotic plaques have provided real-time maps of lipid cores without the need for staining, minimizing processing artifacts and enabling three-dimensional reconstructions of lipid deposits.

The application of lipid histochemistry extends into fields such as oncology, neurobiology, hepatology, and dermatology. For instance, lipid droplet accumulation has been implicated in therapy resistance and metastasis in several cancers, including breast, colon, and prostate cancers. Histochemical identification of lipogenic markers using combined ORO and PLIN2 immunostaining has helped delineate tumor subtypes with distinct metabolic phenotypes [24]. In neurobiology, fluorescent lipid stains have been employed to monitor myelin loss and lipid dysregulation in conditions such as multiple sclerosis and Parkinson's disease.
Moreover, advancements in digital histochemistry and image analysis have enhanced quantification and reproducibility of lipid staining, allowing researchers to employ software-based lipid droplet analysis across large tissue sections and serial slides [25]. This digital integration has not only improved sensitivity but also allowed comparative studies across different experimental models and time points.

Overall, the literature from 2018 to 2025 demonstrates a clear trend toward the convergence of classical histochemical stains with modern molecular and imaging technologies, providing a multidimensional view of lipids in health and disease. The evolution of lipid histochemistry methods reflects both technological innovation and the growing recognition of lipidomics as central to modern biomedical research.

MATERIALS AND METHODS

Search Strategy

This review followed a comprehensive literature search which was conducted across four electronic databases: PubMed, Scopus, Web of Science, and Google Scholar. The search covered studies published from January 1, 2018, to May 2025. Search terms included various combinations and Boolean operators of keywords such as: "lipid histochemistry", "histological staining of lipids", "lipid visualization techniques", "lipid droplet detection", "Sudan stain", "Oil Red O", "Nile Red", "immunohistochemistry and lipids", "CARS microscopy", and "fluorescent lipid staining". Filters were applied to limit the results to peer-reviewed journal articles in English that provided original experimental data or review-level analyses on histochemical methods for lipid detection.

Eligibility Criteria

To ensure that only relevant and high-quality studies were included, a clear inclusion and exclusion criteria was established.

Inclusion criteria

The inclusion criteria included were

i.    Articles published between 2018 and 2025
ii.    Histochemical, immunohistochemical, or imaging-based techniques for detecting or localizing lipids in biological tissues
iii.    Original research or comprehensive reviews with methodological insights
iv.    Detailed protocols, applications, or evaluations of staining or imaging techniques for lipids

Exclusion criteria

Studies were excluded if they:

i.    Focused solely on lipidomics or biochemical extraction methods without histological applications
ii.    Were conference abstracts, editorials, or opinion pieces
iii.    Did not clearly describe the histochemical methodology used
iv.    Were duplicate publications or lacked sufficient methodological detail for evaluation

Data Extraction and Synthesis

All studies meeting the eligibility criteria were extracted, which included data on authorship, year of publication, study design, type of lipid histochemical technique used, tissue or model system studied, methodological protocols, imaging or analysis tools, and key findings related to lipid visualization. Discrepancies between reviewers were resolved through discussion or adjudication by a third reviewer. The synthesis of data was conducted thematically, grouping studies according to the technique used (e.g., classical staining, fluorescence, Raman-based imaging, immunohistochemistry) and their biomedical application (e.g., metabolic diseases, neurological pathology, oncology, developmental biology). This approach allowed us to identify trends, strengths, and limitations across different histochemical methods for lipid detection and to highlight advances in specificity, resolution, and clinical utility.

Quality Assessment

Review articles were evaluated based on the comprehensiveness of their literature inclusion, clarity of methodological analysis, and balance in discussion of strengths and limitations. Each study was scored independently, and those with low methodological quality were excluded from synthesis.

RESULTS

From the systematic search spanning 2018 to 2025, the analysis yielded a broad but thematically coherent body of studies that collectively deepened our understanding of lipid histochemistry. What emerged most clearly was the progressive refinement of techniques that not only detect lipids but also distinguish between their diverse classes—neutral fats, phospholipids, glycolipids, and complex derivatives—within intact tissues. The included studies demonstrated a consistent move toward combining classical staining methods, such as Oil Red O, Sudan Black B, and Nile Red, with modern molecular and spectroscopic techniques. This convergence allowed researchers to address both morphological visualization and quantitative precision, something that had been a limitation in earlier decades.

A striking observation across the reviewed literature was the emphasis on specificity. Traditional lipid stains, while invaluable, were repeatedly shown to lack the resolution to differentiate lipid subclasses. As a result, many recent works turned to complementary methods such as Raman spectroscopy, Fourier-transform infrared (FTIR) imaging, and mass spectrometry–based lipidomics, all of which were integrated into histochemical workflows. These advancements enabled investigators to track lipid accumulation in metabolic disorders, such as non-alcoholic fatty liver disease, with far greater clarity than ever before. The reviewed evidence also highlighted applications in neurodegenerative research, where lipid histochemistry was used to document abnormal sphingolipid and cholesterol deposition in Alzheimer’s and Parkinson’s pathology.

The methodological spectrum also underscored important differences between tissue types. Adipose-rich tissues responded predictably to lipophilic dyes, whereas neural and hepatic tissues often required tailored protocols to minimize artifact formation and lipid extraction during processing. Several studies drew attention to fixation techniques, noting that paraffin embedding frequently led to lipid loss, while cryosectioning preserved native architecture and lipid integrity more reliably. Interestingly, novel cryo-compatible fluorescent probes were reported to produce high-contrast images of lipid droplets without disrupting ultrastructure, bridging a gap between classical histochemistry and high-resolution microscopy.

Another recurrent theme was the shift toward digital quantification. Beyond simple visualization, recent investigations employed image analysis algorithms to measure lipid droplet size, distribution, and density, thus translating histological findings into objective metrics. These digital approaches, when coupled with automated software pipelines, allowed large-scale tissue analyses that reduced observer bias and improved reproducibility. Such developments were particularly significant in metabolic and oncology research, where the heterogeneity of lipid distribution often dictates disease progression and prognosis.

Despite these advances, some differences were evident in the reporting standards across studies. While most papers emphasized methodological optimization, fewer directly addressed inter-laboratory reproducibility, which remains a critical barrier for clinical translation. Moreover, certain lipid classes—particularly glycolipids and specialized signaling lipids—were less consistently studied than neutral fats, suggesting a knowledge imbalance that future research must address.

Overall, the findings of this review reflect a scientific field in transition: moving away from purely descriptive staining toward integrated, multiparametric histochemistry that not only visualizes but also quantifies and functionally interprets lipid biology. The narrative that emerges is one of methodological evolution shaped by the dual demands of precision and clinical relevance.

Table 1. Summary of Results in Lipid Histochemistry (2018-2025)

Table 1. Comparative Overview of Lipid Histochemistry Methods and Applications (2018-2025)

DISCUSSION

The evolution of lipid histochemistry over the past decade has undoubtedly advanced methodological precision and expanded biomedical applications. However, despite these achievements, the field continues to struggle with several unresolved diagnostic limitations and methodological controversies. Classical lipid stains such as Sudan dyes and Oil Red O remain widely used due to their accessibility and reliability in basic research [20,21]. Yet, their persistent use also underscores a broader issue: the limited availability of practical, cost-effective alternatives that offer both high specificity and reproducibility. These conventional dyes lack the ability to discriminate between lipid subclasses, which continues to hinder nuanced pathological interpretations, particularly in diseases where distinct lipid species carry different biological or diagnostic implications.

The recent revival of frozen-section staining with lipophilic dyes such as Oil Red O and Nile Red reinforces this tension. Although these dyes remain effective for detecting neutral lipids in metabolic models such as hepatic steatosis, their inability to distinguish finer lipid profiles (e.g., cholesterol versus cholesteryl esters) remains a critical diagnostic limitation [22]. Even where they are considered “gold standard” techniques, such reliance exposes an unresolved gap: the lack of a universally accessible histochemical method capable of both rapid visualization and subclass-level discrimination.

Advanced vibrational microscopy techniques like CARS and SRS have attempted to fill this gap, offering label-free, high-resolution lipid imaging with some subclass specificity. However, their adoption remains limited by high cost, technical complexity, and inconsistent standardization across laboratories. While intraoperative SRS imaging shows promise for real-time tumor margin assessment [23], the technique raises questions regarding sensitivity thresholds, inter-observer variability, and the reproducibility of vibrational signatures between tissue types. These unresolved challenges continue to delay their transition from research tools to routine diagnostic instruments.

Similarly, the use of lipid-specific immunohistochemistry (IHC) targeting molecules such as perilipins, adipophilin, and apolipoproteins has improved cellular localization of lipid droplets in diverse tissues [24]. Yet, antibody-based lipid detection is inherently indirect, inferring lipid presence from associated proteins rather than visualizing lipids themselves. This limitation becomes especially problematic in conditions where lipid droplets are structurally abnormal or protein-coating patterns change, such as neurodegenerative diseases [25]. The increasing reliance on hybrid approaches, such as MSI integrated with IHC, reflects an acknowledgment that no single method currently offers a complete spatial and molecular picture.

One of the most significant ongoing controversies concerns the detection of complex lipids like gangliosides, ceramides, and phosphatidylinositols which often evade dye-based histochemical methods due to their amphipathic and chemically unstable nature. MSI, MALDI, and DESI have revolutionized the mapping of such lipids in tissues [26], but these techniques also suffer from ionization variability, analyte suppression, and a lack of universally accepted calibration standards. These limitations not only complicate inter-study comparisons but also restrict clinical deployment.

Tissue preservation remains another unresolved challenge. Paraffin embedding, although routine in clinical pathology, extracts most lipids during dehydration, rendering paraffin sections unsuitable for classical lipid staining; meanwhile, frozen sections preserve lipids but compromise tissue morphology and long-term storage quality. Attempts to reconcile these opposing constraints such as cryoembedding with high-pressure freezing or resin infiltration show promise, but lack scalability and are rarely implemented in clinical laboratories, limiting their broader diagnostic utility.

CONCLUSION

This review shows that while classical stains such as Sudan dyes and Oil Red O remain important for routine and translational research, they are now increasingly supported by higher-resolution and more molecularly informative methods, including Nile Red fluorescence, FTIR, and MALDI-MSI. These tools have improved sensitivity, allowed better subclass discrimination, and enabled more precise spatial mapping of lipid alterations across diverse tissues. A consistent trend across studies is the movement toward multimodal imaging, combining optical, chemical, and immunohistochemical techniques to generate more comprehensive lipid profiles in conditions such as cancer, neurodegeneration, and metabolic disease.
Despite this progress, significant challenges persist. Variability in tissue preparation, limited standardization across laboratories, and restricted accessibility of advanced imaging platforms continue to hinder reproducibility and slow clinical adoption. Additionally, although digital pathology and machine learning show promise in improving lipid quantification, validated workflows remain scarce.

ACKNOWLEDGMENTS

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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