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Review

Histological and Immunolabeling Techniques in Arabidopsis thaliana: A Practical Guide and Standardization Roadmap

by
Samuel Valdebenito
1,
Alexis Rubio
2,
Alejandra Moller
2,
Javier Santa Cruz
3,
Priscila Castillo
2,
Mayra Lirayén Providell
2,
Camila Cáceres
2,
Diego Calbucheo
4,
Ignacia Hernández
5 and
Patricia Peñaloza
3,*
1
Escuela de Ciencias Agrícolas y Veterinarias, Universidad Viña del Mar, 2520000 Viña del Mar, Chile
2
Escuela de Tecnología Médica, Universidad de Valparaíso, 2520000 Viña del Mar, Chile
3
Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, 2260000 Quillota, Chile
4
Laboratorio de Ecoinformática, Instituto de Ciencias Ambientales y Evolutivas, Universidad Austral de Chile, 5090000 Valdivia, Chile
5
Departamento de Producción Vegetal, Facultad de Agronomía, Universidad de Concepción, 4030000 Concepción, Chile
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(10), 2357; https://doi.org/10.3390/agronomy15102357
Submission received: 10 September 2025 / Revised: 28 September 2025 / Accepted: 30 September 2025 / Published: 8 October 2025
(This article belongs to the Collection Propagation and Conservation of Horticultural Plants: In Vitro and In Vivo)
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Abstract

Arabidopsis thaliana is a widely used model in plant biology, where histology (HT), histochemistry (HC), immunohistochemistry (IHC), and immunofluorescence (IF) are applied to study cellular structures, macromolecules, and antigens. Despite their extensive use, protocols lack standardization and exhibit substantial variability in critical aspects such as reagent handling, exposure times, and the proper use of controls. This methodological heterogeneity represents a major gap, limiting reproducibility and comparability between studies. Unlike previous methodological reviews, this work focuses exclusively on A. thaliana, systematically identifies reporting omissions, and proposes a roadmap for standardization. A narrative review of literature retrieved from Scopus and Web of Science was conducted with the aim of analyzing methodological approaches, identifying inconsistencies, and offering recommendations for improved laboratory practices. The analysis revealed frequent omissions in the reporting of critical steps such as dehydration, clearing, antigen retrieval, enzyme blocking, and the incorporation of positive and negative controls, which compromise the reliability of results and hinder inter-laboratory validation. Based on this evidence, three key recommendations are emphasized: (i) organ-specific selection and explicit justification of fixatives and stains; (ii) mandatory incorporation of positive and negative controls in IHC and IF; and (iii) adoption of a minimum reporting checklist to enhance reproducibility. Beyond cell morphology, the reviewed studies demonstrate applications in plant physiology, phytogenetics, and pathophysiology. By combining critical analysis with actionable guidelines, this review contributes a practical reference to strengthen methodological rigor in histological and immunological studies of plants.

1. Introduction

A. thaliana is an angiosperm belonging to the Brassicaceae family. It is widely used as a model organism in scientific research [1,2,3,4,5] due to its short life cycle of approximately 5 to 6 weeks [4], and its fully sequenced genome, which facilitates its use in various scientific disciplines [2,3,6,7,8]. This species plays a crucial role in studies across fields such as plant biology, genomics, molecular biology, and histology. Within these investigations, techniques such as histology, histochemistry, immunohistochemistry, and immunofluorescence are applied; these methods are essential for both qualitative and quantitative studies at the morphological, tissue, and cellular levels.
The histological technique (HT) allows for the anatomical description of structures [9,10,11,12,13,14,15,16] as well as their functional and phytopathological study [17,18,19,20,21]. This is possible through the staining of samples, given that most tissues lack inherent coloration, making the application of dyes essential for the identification and differentiation of structures [22]. In this context, the routine Safranin-Fast Green (S-FG) staining is widely used for plant histological studies. A comparative summary of the main staining techniques, their chemical nature, and their applications in animal and plant tissues is presented in Table 1.
Histochemistry (HC), on the other hand, focuses on the chemical study of tissues, allowing for the identification and localization of organic macromolecules via reactions that color them, rendering them visible under an optical microscope [23]. Its application is common in studies examining physiological responses and biomolecule variation [24,25,26,27,28]. The most commonly used dyes include Sudan and PAS [29,30,31,32,33,34,35,36,37].
HC is more specific than HT since it allows for the identification of subcellular compounds with greater precision. Histochemical identification in plant tissues requires a rigorous standardization of the method for each species due to the variability among them [22,23].
Immunohistochemistry (IHC) and immunofluorescence (IF) techniques are molecular interaction methods that enable the in situ identification of cellular and extracellular components (antigens) using specific antibodies, detection systems, and visualization methods [38,39,40,41]. In IHC, visualization is achieved via the antigen–antibody interaction, employing common methods such as Streptavidin-Biotin (EAB) and Peroxidase-Anti-Peroxidase (PAP) [42,43,44,45,46,47]. In IF, identification is carried out by the binding of the antigen to a specialized fluorochrome [48,49,50,51,52,53]
The development and proper application of these techniques in plant research require specialized skills from analysts, as well as precision in laboratory procedures. A lack of proficiency in aspects such as reagent handling, reaction times, and methodological fundamentals can lead to incomplete or erroneous results.
Unlike previous methodological reviews, this work focuses exclusively on A. thaliana, systematically identifies reporting gaps in published protocols, and proposes a practical reporting checklist to guide standardization. Therefore, the objective of this narrative review was to analyze methodological procedures in HT, HC, IHC, and IF, and to recommend precise adjustments aimed at improving reproducibility and cross-laboratory comparability. In particular, this review emphasizes modifications tailored to different plant organs and provides updated guidelines for methodological rigor in recent A. thaliana studies.
This work is structured into five sections: first, the processing of the sample; second, the specific processing for HT and HC; third, IHC and IF; and in the final two sections, the study objectives, areas of investigation, and recommendations are discussed.

2. Stages of Processing Plant Samples

2.1. Fixation and Dehydration

Histological processing involves a series of steps that take plant tissue from fixation to paraffin infiltration, as shown in Figure 1. Among the studies reviewed, glutaraldehyde was frequently used as a fixative, primarily recommended for electron microscopy due to its excellent ability to preserve ultrastructure. However, this fixative is not suitable for detecting aldehyde groups, as it can lead to overstaining caused by the Schiff reagent reacting with the fixative’s chemical groups bound to the tissue [22], potentially resulting in overestimation of target structures. Despite this, several studies employing the PAS technique [33,54,55,56] used glutaraldehyde, which represents a methodological error. Only [33] justified its use, as the same samples were prepared for transmission electron microscopy. None of the analyzed studies included positive or negative controls, increasing the risk of false-positive interpretations.
Regarding fixative mixtures, the use of FAA and Carnoy was reported. FAA, composed of formaldehyde, acetic acid, and ethanol, is suitable for preserving both lignified and non-lignified tissues, while Carnoy, being nonpolar, is effective in preserving sugars [22,57]. Nevertheless, the reviewed studies did not provide a rationale for their choice of fixative, using them only in alignment with the primary experimental design, without a critical discussion of their suitability for the intended staining procedures.
The dehydration process is performed using an ascending series of alcohol solutions, ranging from 30% alcohol to absolute alcohol (100%), with the goal of completely removing water from the fixed tissues so that clearing can then be carried out [22].
The studies analyzed do not specify dehydration times [58,59,60,61,62,63,64,65,66,67]. However, an approximate duration of four days is recommended to ensure proper penetration of solvents into plant tissues, beginning with a 30% alcohol concentration and gradually progressing to absolute alcohol [57,68]. This careful process is necessary to avoid damaging cellular and tissue structures due to sudden concentration changes. The prolonged duration is attributed to the presence of the plant cell wall, which acts as a barrier between the exterior and interior of the cell, restricting solvent permeability to the cytoplasm and consequently slowing the dehydration process [22].
For clearing (or diaphanization), a polar solvent is employed to facilitate the transition from polar alcohol to apolar paraffin, replacing the dehydrating agent with a clearing liquid that renders the sample translucent and enhances its microscopic visualization [57]. Most of the analyzed studies do not detail the clearing process or mention the solvent used. This step is crucial in histology, so it is inferred that studies using conventional microscopy and fluorescence likely performed this procedure. In contrast, electron microscopy studies used propylene oxide as the clearing agent [69].
To achieve proper dehydration and clearing, it is recommended to perform an ascending series of alcohol and xylene in ratios of 3:1, 1:1, and 1:3, with each step lasting two hours, and then finishing with pure xylene overnight. This technique ensures a gradual and slow penetration of solvents into the tissues [57,68]. An optimized dehydration and infiltration protocol adapted from [68] is summarized in Table 2.

2.2. Impregnation

Impregnation is the process that follows clearing, where tissues are immersed in a liquid that penetrates them deeply, preparing them for embedding in a solid block to facilitate subsequent sectioning [70]. The most commonly used solvent is paraffin, a mixture of hydrocarbons with a melting point range between 45 °C and 60 °C [71]. In histological techniques, paraffin is typically used at temperatures between 54 °C and 58 °C, whereas in immunohistochemistry, lower temperatures, between 45 °C and 50 °C, are employed to prevent the denaturation of epitopes [70].
Epoxy resin is the second most used substance in the impregnation process. It is composed of a poly(aryl-ether) monomer of glycerol, which contains two terminal epoxy groups (oxygen atoms bonded to carbon atoms), along with polyamine and/or acid anhydride, which generate cross-linking bridges that provide significant hardening power [57,70,71]. This resin is primarily used in electron microscopy, as it offers greater support for sectioning ultra-thin tissues (at the nanometer level) and better preserves the cellular ultrastructure compared to paraffin [72].
Both paraffin and epoxy resin have been used to observe tissue and cellular structures in greater detail [33,54,55,73]. These procedures align with previously described impregnation protocols [22,71]. Although specific details regarding solvent times and concentrations used during these stages were not provided in the reviewed studies, an ascending series of xylene-paraffin, as mentioned for the dehydration stage, is recommended.

2.3. Cutting Instrument and Section Thickness

The instruments used in the studies include both microtomes and ultramicrotomes. It is important to note that the section thickness of a histological sample depends on factors such as tissue type, the microscope employed, and the staining technique used for visualization [57]. For microtomes, a section thickness ranging from 0.5 to 10 μm is recommended, as this range permits sufficient light transmission through the tissue for optimal observation with an optical microscope [45,57,71]. In contrast, ultramicrotomes produce sections between 20 and 80 nm thick, which offers enhanced resolution for electron microscopy [72].
The results show that the average section thickness is approximately 6.6 μm, with values ranging from 3.2 to 11.4 μm [58,59,60,61,62,63,64,65,66,67]. Notably, the thickest sections—measuring around 11.4 μm—were used for Toluidine Blue staining. This can be explained by the fact that the authors [74,75,76,77,78,79] did not intend to observe protein details or subcellular structures, but rather the overall tissue architecture, such as xylem and phloem.
In contrast, immunohistochemistry and immunofluorescence studies (IHC-IF sections with an average thickness of 3.2 μm aimed to analyze finer details of the tissue, such as the cytoplasm and plasma membrane [43,44,55,80,81,82,83].

3. Specific Processing by Techniques: Protocol Description

3.1. Hematoxylin–Eosin (H-E)

Hematoxylin, being a cationic compound, stains acidic or negatively charged structures—such as nucleic acids—resulting in a purple color. In contrast, eosin is an anionic dye that stains basic structures, such as the cytoplasm and extracellular matrix, imparting a pink hue [22,57]. In plant tissues, the reviewed studies [84,85,86,87] report that nuclei are stained purple, yet the differentiation of lignified structures is not fully achieved since this staining colors both the plant cytoplasm and cell walls pink.
However, these data are well established in the literature for their use in animal tissues, where hematoxylin staining times vary between 3 and 5 min, and eosin only requires 15 s [22,57,68,70]. Regarding the concentration and preparation of hematoxylin, it is recommended to prepare it at 1% in absolute alcohol, then dilute it 20-fold in an aqueous solution with ammonium alum. Subsequently, mercuric oxide and acetic acid are added, although this mixture is now commercially available. On the other hand, eosin is prepared at 0.5% in absolute alcohol, although it can also be found pre-prepared on the market [22]. The collected studies do not detail the methodology used in H-E staining, making it difficult to determine whether the observed variability in some published studies is due to the procedure, staining time, or dye concentration.
In some studies [85,86,87] variability in cell type differentiation is evident; however, in others, like [84] detailed cellular types, nuclei, and overall morphometry are described, highlighting the difficulty posed by the omission of staining times and solution concentrations.
Therefore, in the context of the reviewed studies, hematoxylin does not appear to be a specific stain for observing lignified structures, nor does it fulfill a routine role comparable to that it plays in animal tissues. Its utility in plant tissues lies primarily in identifying cell division and growth, as well as nuclear details [57]. This distinguishes it from other techniques, such as Safranin–Fast Green (S-FG) and Toluidine Blue, which have proven to be more effective in the observation of lignified structures [65,66,76,88,89,90].

3.2. Safranin–Fast Green (S-FG)

Safranin is applied for 30 min to stain anionic lignified structures, such as sclerenchyma and xylem [68]. The prolonged use of Safranin in plant tissues is part of a dual-staining protocol based on differences in solubility affinity: first, Safranin (which is highly soluble in alcohol) is applied, followed by Fast Green (also prepared in alcohol). The longer the tissue is exposed to Safranin, the less it will be decolorized by the subsequent Fast Green treatment, thereby enabling more precise staining [57,70].
This technique is used as a routine stain for plant tissues [58,59,60,62,63,64,65,66,67,90,91], as it allows for the general differentiation between lignified and non-lignified structures. In cases where a more detailed histological analysis is required, other techniques such as PAS, Sudan, IHC, or IF may be employed [38,39,71].
The technical details reported in the reviewed studies [58,59,60,62,63,64,65,66,67,90,91] indicate that the concentration of both Safranin and Fast Green is 1%—the former in a semi-alcoholic solution and the latter in an alcoholic solution. This is consistent with the description provided by [68]. With regard to staining times, only [57] is cited, recommending a 2 h staining for Safranin and 15 s for Fast Green. However, these times differ from the range recommended by [68], who suggests 30 min for Safranin and 2 to 15 s for Fast Green.

3.3. Toluidine Blue

Toluidine Blue is a cationic dye widely used due to its unique properties. Its ions exhibit different wavelengths depending on the tissue with which they interact; if the tissue appears blue, the dye is considered orthochromatic, whereas if it appears reddish or purplish, the effect is metachromatic—a phenomenon known as metachromasia. Additionally, because it is a rapid, single-step staining procedure, it is especially advantageous for laboratory applications [22,57].
Several studies have reported a concentration range of 0.02% to 0.05% [74,75,76,77,78,79,88,89,92,93,94,95,96]. This concentration does not match that used in animal tissues, which is typically 0.5% [22]. The difference may be attributed to the cationic nature of the dye and its higher affinity for acidic components such as lignin, as well as for basic components such as the cytoplasm and primary cell wall in plant tissues. This likely explains why plant tissues retain more dye compared to animal tissues.
Regarding staining times, the studies indicate a range between 30 and 45 s, which falls within the literature-recommended range of 1 s to 3 min. The methodology described by the authors appears appropriate, as it allows for clear differentiation of important plant structures, such as xylem and phloem.
Based on the above, both Safranin–Fast Green (S-FG) and Toluidine Blue are widely used staining techniques in plant tissues. However, Toluidine Blue offers some advantages over S-FG. Among these, its simple preparation stands out—requiring only one staining step instead of two—and a shorter application time. Additionally, the metachromatic nature of Toluidine Blue allows for a clearer differentiation of plant structures: the parenchyma appears purple, the phloem stains reddish, and the xylem exhibits blue tones [68]. These characteristics suggest that Toluidine Blue could be considered a preferred option for histological studies in plant tissues, particularly in those cases where rapid and clear structural differentiation is desired.

3.4. Periodic Acid–Schiff (PAS)

The PAS staining is a histochemical method that identifies carbohydrates. The first step involves oxidizing aldehyde groups with periodic acid; these groups are then detected with Schiff’s reagent, producing a magenta color [57,71].
The reviewed studies [33,54,55,56,73,81,97,98,99,100,101,102] indicate that the concentration of periodic acid varies between 0.5% and 1%. Regarding application time, only three of the twelve reviewed studies mention exposure times ranging between 5 and 30 min. However, the literature advises that the exposure to periodic acid should not exceed 10 min [22,57] as longer times can oxidize other hydroxyl groups, potentially resulting in non-specific staining and false positives.
For the application time of Schiff’s reagent, studies report durations varying between 20 and 60 min at a concentration of 0.5%. In animal tissues, the literature recommends an exposure time of 30 min for Schiff’s reagent [22,23,57,70]. Prolonged oxidation and staining times can lead to saturation of the aldehyde groups, also causing non-specific results. This effect may be mitigated by using sulfurous water [22].
An important point to note is that none of the reviewed studies explicitly mentions the use of external positive controls for the PAS technique. The absence of these controls could lead to misinterpretation of the results and the generation of false positives. If such controls were performed but not reported, there is reasonable doubt regarding the methodological rigor of the work. Nonetheless, some studies, such as those by [54,81], present high-quality morphological results, for example, the identification of PAS-positive starch granules in reserve parenchyma. In these cases, it is suggested that, to ensure the validity of the results, the use of external positive controls is necessary.
Ref. [97] report certain staining errors, as the tissue morphology is not clearly discernible in the images. In plant tissues such as leaves, it would be expected that starch granules in the reserve parenchyma stain purple, and that cell walls are distinctly delineated features, which were not observed in that study [22].

3.5. Sudan

Sudan corresponds to a lipid-soluble lisochrome dye used for the histochemical identification of lipids. These substances dissolve in graduated alcohols and are then transferred to the lipid deposits in tissues [28].
In the reviewed studies [98,103,104,105,106,107,108,109,110,111,112], Sudan concentrations vary between 0.05% and 3%, using alcohol as the solvent in percentages ranging between 70% and 92%. In contrast, the literature on animal tissues [22] describes a standard concentration of 0.5% in 70% alcohol and an application time of 60 s. In plant studies, staining times range from 30 s to 2 h; however, the reported optimal times vary between 8 and 10 min with concentrations around 2%, as shown in the study by [105], which demonstrated improved staining of the endodermis in plant roots.
The variation in staining times can be attributed to the nature of lipids in plants, which are primarily composed of cutin and suberin; these substances are made up of hydroxylated and epoxidized fatty acids [113,114,115] linked by long, branched ester bonds, making them more complex molecules and consequently more difficult for the dye to solubilize [22]. Therefore, it is advisable to extend the staining times in plant tissues to ensure adequate visualization of these macromolecules.
Regarding fixatives, formaldehyde was most commonly mentioned in the studies, which is consistent with [22] description, as this fixative prevents the lipids from dissolving due to its intrinsic polarity. Additionally, it is recommended to add calcium to the fixative solution to stabilize the tissue lipids [57].
In studies using fresh tissue, the cryostat technique is most often employed, as it uses liquid nitrogen to maintain cellular integrity and prevent lipid loss during the solvent baths. However, this technique has the limitation that tissues cannot be preserved for extended periods for observation, as they degrade more rapidly [57].

3.6. Specific Processing for IHC and IF

In the reviewed studies, the most used fixatives for immunohistochemistry (IHC) and immunofluorescence (IF) were paraformaldehyde and glutaraldehyde [55,80,81,105,116]. This is in accordance with the recommendations by [38,39,117] who suggest the use of paraformaldehyde at concentrations between 2% and 4%, with the addition of glutaraldehyde at concentrations between 0.1% and 0.4% to enhance tissue morphology preservation. Figure 2 provides a schematic overview of the sequential steps involved in IHC and IF for A. thaliana, highlighting critical stages such as fixation, antigen retrieval, enzyme blocking, and the use of appropriate controls. This visual reference complements the methodological description and underscores the importance of protocol standardization.
Regarding antigen retrieval, none of the collected studies detailed this procedure in their methodologies. Concerning the blocking of endogenous peroxidase, this enzyme in plant tissues has functions similar to those in animal tissues, but also additional roles, such as xylem differentiation and protein assembly in the cell wall [38,39,41,117]. Blocking endogenous peroxidase is essential to avoid false positives in IHC, as the activity of this enzyme can generate unreliable results. To deactivate it, hydrogen peroxide is used, as performed in the reviewed IHC studies [82,118,119], with concentrations ranging between 0.03% and 3%; however, the majority of the studies did not specify the application times.
For blocking non-specific binding sites, most studies [55,80,81,82,105,118,120] used fetal bovine serum at 1% in PBS, which is consistent with IHC protocols for animal tissues, suggesting that this step is equally necessary in plant tissues. However, only one study [119] mentioned the use of negative controls with PBS, while the remaining studies did not detail this aspect.
It is crucial that IHC and IF studies include controls and proper antigen retrieval, as these ensure correct antigen labeling. Studies that included controls, such as [119] demonstrated better labeling quality, as evidenced by the expression of AVP1 in the phloem. In contrast, studies like [118] that did not mention controls showed less reliable labeling.
In IF studies [48,52,55,80,81,83,105,116,120,121,122,123,124,125,126] adequate labeling is observed, with target structures clearly differentiated from non-target structures—for example, the labeling of cell wall components with LM15 in xylem tissues.
Regarding signal amplification systems in IHC, methods such as APAAP (Alkaline Phosphatase Anti-Alkaline Phosphatase) and PAP (Peroxidase Anti-Peroxidase) were employed. In studies using APAAP [118] no blocking of alkaline phosphatase was performed, which could have generated false positives. In contrast, studies employing PAP [82,119] included the appropriate blocking step, resulting in improved labeling—suggesting a preference for PAP over APAAP.
Concerning the chromogens and fluorochromes used, DAB and AEC were the most common [82,118], with better labeling quality observed compared to NBT-BCIP, which produced more diffuse results [119]. Studies also employed fluorochromes such as Alexa Fluor 488, Cy2, and Cy3, which provided clear labeling and differentiation of target structures [81,105,121,122,123,124].

4. Focus of Studies: Cellular Morphology, Cell Differentiation, Detection of Suberin, Lignin, and Sugars, Cell Growth, Cell Death, and Protein Expression

In the reviewed studies, the primary objective is the observation of cellular morphology, as shown in Figure 3. This is present in various research areas, such as plant physiology, where morphology is involved in observing the development of organs during plant growth [52,60,64,73,86,92,93,99,101,124], and also in morphological changes resulting from agricultural practices [73,88,101]. Consequently, in some cases, hormonal, nutritional, and phenological changes are also examined. Other studies aim to observe changes in the cellular parenchyma of plants under water stress [4,7,58,105,110], which is especially relevant in the current climate crisis, where both agricultural and botanical fields are searching for plant varieties resistant to drought, flooding, and vegetative stress. In the search for resistant varieties, cellular morphology must also be evaluated [60,93]. Additionally, some studies focus on phytopathology, i.e., demonstrating damage caused by fungi, viruses, and bacteria in comparison to a normal plant [65,77,78,87,95,118]. Techniques used to achieve these objectives include Safranin–Fast Green, Toluidine Blue, and PAS.
Regarding studies aimed at evidencing cell differentiation, their applications include observing the transformation of totipotent cells into male gametes [84], into vascular tissues [59,66,75,82,119] and the effect of hormones on this process [81,105]. Practically, the techniques used for this objective were S-FG, PAS, and IHC–IF, applied in both agronomic and botanical fields. These techniques allow the visualization of the effects of agricultural practices, including exposure to physical factors (temperature, humidity, and soil compaction resistance), chemical agents (hormones, fungicides, growth factors), and biological agents such as pathogens and pests.
Among the analyzed studies, suberin stands out. It is a polymer composed of hydroxylated and epoxidized fatty acids linked by ester bonds, forming long, branched chains [104]. This molecule functions as a protective barrier against external factors such as temperature, humidity, and pathogens [127] and is found in many plant organs. Its location and function are relevant in both physiological and pathological contexts. The absence or deficiency of suberin is also of interest to botanists and plant pathologists. This molecule was the subject of various studies, where it was observed as a lipid protecting the root [103,104,110,111] confirming its protective role in the stem, root, and leaf against external agents [105,106,107] as well as its relevance in evaluating fatty acid production potential [109] under physical, chemical, and biological stimuli. In practice, Sudan III and IV are the main techniques used to detect this macromolecule, which is valuable for assessing the thickness and presence of this lipid in the context of protection against drought, extreme temperatures, and pathogens in crops of economic interest [128]
Lignin is a heteropolymer present in the cell walls of plant vascular tissues, providing structural rigidity and resistance to mechanical stress and water pressure. Its identification is critical for distinguishing lignified from non-lignified cells, providing researchers with insights into the general architecture of the tissue under study. The reviewed studies aimed to identify morphological structures in lignified tissues [62,67,76,79], hormones associated with lignification, and genes involved in this process [62].
The importance of identifying this natural polymer lies in its role as one of the main components of biomass for biofuel, paper, and cork production [129]. Thus, S-FG and Toluidine Blue techniques, including their application in A. thaliana, contribute to understanding the localization and distribution of this industrially valuable molecule. With these stains, lignin appears red and violet, respectively.
Regarding starch detection, the reviewed studies aimed to observe the presence of starch granules [54], vascularization [97] inhibition of chloroplast formation and its effect on starch production [104], and the importance of starch-degrading enzymes [33,56]. In this context, PAS staining is useful for examining the effects on primary plant metabolism, specifically sugar dynamics under the influence of physical, chemical, and biological agents [127,128] and it can also be applied to other plant species.
Cell growth was another detailed research objective, with studies evaluating changes in organ cell size and their development [84,100,112], detecting modifications in amyloplasts [55,90,94], observing cellular injuries [83], and localizing protein expression and its impact on cell growth [85,116,119]. In these cases, genomic or environmental factors influence the distribution and presence of sugars and lipids. From a practical perspective, cell growth can be analyzed using H&E and S-FG techniques, and based on the results in A. thaliana, these methods could be valuable in describing physiological phenomena in edible plants and crops of agronomic interest.
Cell death was also a key objective in the analyzed studies. Morphological changes and necrosis caused by bacterial action were documented [65] as well as responses in transgenic plants and the effect of acetylation on the expression of fungal defense mechanisms—showing cell death in areas without defense activation [77]—and the presence of cellular markers during pathogen infections [116]. S-FG and IHC–IF techniques are useful for analyzing plant cell death when pathogens affect agriculturally or industrially important organs. S-FG allows the observation of tissue discontinuities and necrosis [77,118], while IHC–IF permits the detection of markers related to cell renewal and death [87,116].
In studies focused on cellular expression, various molecular markers related to normal and pathological plant metabolism were identified, including Anti-PIN1, GYM5, GFP, Anti-β-tubulin, ATNRAMP4, Anti-ABA, JIM, Anti-AVP, LM10, and LT130. The techniques used to visualize these markers were IHC–IF, which can be applied to other species when monitoring plant responses to stress conditions [6,48,55,81,107,118,125,126]. These stressors, whether physical, chemical, or biological, can alter the organism’s physiology, making it necessary to localize molecular responses with precision.

5. Recommendations for Protocol Application

Based on the studies reviewed, the following recommendations are made to improve HT (histological techniques) and HC (histochemical techniques) in the microscopic observation of plant organs:

5.1. Flower

Hematoxylin–Eosin (H&E) and Periodic Acid–Schiff (PAS) techniques are commonly used, as they allow for the identification of cell growth and differentiation. The flower is an area of high cell division [127]. Fixation for this organ is generally carried out using Formalin–Acetic Acid–Alcohol (FAA), and for improved structural detail, a buffered solution is suggested [22], as it better preserves cytoarchitecture. The preferred cutting instrument is the microtome, with an average section thickness of 10 microns

5.2. Leaf

Safranin–Fast Green (S-FG) and PAS techniques are recommended. The former allows for the identification of cell morphology and vascular bundles, which are important structures in the leaf [68]. PAS is suitable for visualizing starch granules in the parenchyma of this organ. FAA is recommended as the fixative for optimal histological and histochemical visualization. The suggested cutting instrument is the microtome, producing sections of approximately 7.6 microns in thickness.

5.3. Root

Sudan staining is frequently used to detect lipids inside and outside the root, and Toluidine Blue to distinguish lignified from non-lignified structures [94,97]. When rapid sectioning is required, or for lipid detection, the use of a cryostat is recommended; for general structural identification, Toluidine Blue is preferred, using cuts with an average thickness of 10 microns.

5.4. Seed

PAS and S-FG techniques are commonly used. PAS is useful for identifying starch reserves in the storage parenchyma of the embryo; FAA is the ideal fixative for this stain, while glutaraldehyde should be avoided due to the risk of false positives [22]. S-FG is useful in seeds for identifying the palisade parenchyma in the cotyledons, the cellular structure and division in the radicle, and meristematic details that will generate the aerial structures of the plant [127]. The recommended cutting instrument is the microtome, with an average section thickness of 6.9 microns.

5.5. Stem

S-FG is recommended for identifying stem structures, as it allows observation of both lignified and non-lignified tissues, including shoot meristems, pith parenchyma, and epidermis [68]. For fixation, FAA and glutaraldehyde are ideal, depending on the required cellular detail. The cutting instruments used by the authors include both the ultramicrotome and the microtome, producing sections with an average thickness of 5.5 microns.

5.6. Considerations Regarding Section Thickness

Despite differences in section thicknesses among the reviewed studies, the resulting images do not show significant differences in quality. However, there are no clear technical justifications for the variation in section thickness for each organ. For instance, in animal histology, section thickness depends on the number of cells in the tissue—the greater the number, the thicker the section [22]. In plant cells, which are generally larger, a greater section thickness may be required for optimal visualization [68]. Therefore, it is recommended to use the thicknesses indicated by the authors, ranging from 5.5 to 10 microns.

6. Conclusions

Histological, histochemical, immunohistochemical, and immunofluorescence techniques are essential tools for the detailed study of plant tissues in A. thaliana, enabling significant advances in areas such as plant physiology, phytogenetics, and phytopathology.
Proper fixation is fundamental for preserving plant cytoarchitecture. Fixative mixtures such as Carnoy and FAA have proven effective in this regard. It is crucial to select the appropriate fixative depending on the technique and the objective of the study.
The routine staining techniques evaluated, such as Safranin–Fast Green (S-FG) and Hematoxylin–Eosin (H-E), are useful for identifying morphological structures and observing cell growth in plant tissues. Toluidine Blue offers significant advantages as a routine stain due to its metachromatic properties, ease of preparation, and shorter staining times, allowing for faster and more accurate identification of cellular structures.
The histochemical techniques PAS and Sudan are valuable for detecting specific macromolecules such as polysaccharides and lipids in plant tissues. However, it is necessary to standardize their application protocols in plants, adjusting times and concentrations, and ensuring the use of both positive and negative controls to guarantee the precision and reproducibility of the results.
Immunohistochemistry (IHC) and immunofluorescence (IF) are effective tools for studying the expression and localization of proteins in plant tissues. Their application requires strict standardization of protocols, including critical steps such as antigen retrieval, blocking of endogenous enzymes, and proper use of controls. The appropriate selection of detection systems is essential to avoid nonspecific results and to ensure the reliability of the data obtained.
It is imperative to develop standardized and tissue-specific technical protocols for plant tissues in all the techniques mentioned. Additionally, improving the standardization and detailed documentation of procedures will increase the reproducibility and validity of the results, bringing plant histology practices to the level of rigor seen in animal histology.
Although this study focused on A. thaliana, the findings and recommendations are applicable to a wide variety of plant species, opening opportunities for future research in comparative histology and expanding knowledge in plant sciences. It is recommended to explore and adapt other histological, histochemical, immunohistochemical, and immunofluorescence techniques used in animals to the study of plant tissues, which could reveal new aspects of plant biology.
Unlike previous methodological reviews, this work focuses exclusively on A. thaliana, synthesizes methodological gaps, and provides practical comparative tables and optimized protocols. By emphasizing reproducibility, cross-laboratory comparability, and transparent reporting, this review contributes a unique roadmap for standardization in plant histology and immunolabeling.

Author Contributions

Conceptualization: S.V., P.P., A.M., and A.R.; literature search and data curation: J.S.C., D.C., C.C., and I.H.; resources: S.V., P.P., and A.M.; supervision: P.P.; visualization: A.R., D.C., and C.C.; writing—original draft preparation: S.V., A.R., D.C., and P.C.; writing—review and editing: P.P., J.S.C., A.M., and M.L.P. All authors have read and agreed to the published version of the manuscript.

Funding

Javier Santa-Cruz (J.S.C.) and Samuel Valdebenito (S.V.) were awarded funding through the InES I + D initiative of the Vicerrectoría de Investigación, Creación e Innovación at the Pontificia Universidad Católica de Valparaíso (project PUCV TPI 02-INID230010). J.S.C. and S.V. were supported by ANID MSc scholarships (No. 22231615 and No. 22241938, respectively). In addition, J.S.C. received funding from project ND-26, financed through the Tesis para Impactar el Territorio program by Nodo CIV-VAL, as well as from the Match Maker program of the Dirección General de Vinculación con el Medio at PUCV.

Data Availability Statement

No new data were created or analyzed in this study.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Figure 1. Stages of processing plant samples.
Figure 1. Stages of processing plant samples.
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Figure 2. Specific processing for IHC and IF.
Figure 2. Specific processing for IHC and IF.
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Figure 3. Integrated diagram summarizing the main research areas and cellular objectives studied in A. thaliana. The scheme highlights processes such as growth, development, differentiation, morphology, and cell death, and the techniques applied to investigate them, including histological, histochemical, immunohistochemical, and immunofluorescence methods.
Figure 3. Integrated diagram summarizing the main research areas and cellular objectives studied in A. thaliana. The scheme highlights processes such as growth, development, differentiation, morphology, and cell death, and the techniques applied to investigate them, including histological, histochemical, immunohistochemical, and immunofluorescence methods.
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Table 1. Staining techniques, chemical nature, and target zones in animal and plant tissues. Comparison of classical histological stains originally developed for animal histology and their adaptation to plant tissues, with emphasis on applications in A. thaliana. The table summarizes the chemical nature of each dye, the main structures stained in animal versus plant tissues, and their most frequent applications in histological and histochemical studies. Compiled and adapted from [22,23], with modifications by the authors.
Table 1. Staining techniques, chemical nature, and target zones in animal and plant tissues. Comparison of classical histological stains originally developed for animal histology and their adaptation to plant tissues, with emphasis on applications in A. thaliana. The table summarizes the chemical nature of each dye, the main structures stained in animal versus plant tissues, and their most frequent applications in histological and histochemical studies. Compiled and adapted from [22,23], with modifications by the authors.
TechniqueChemical NatureAnimal Tissue StainingPlant Tissue StainingApplications
Hematoxylin-Eosin H: Cationic/E: AnionicH: Nuclei/E: CytoplasmH: Nuclei/E: CytoplasmGeneral structural staining
Safranin-Fast GreenS: Cationic/FG: AnionicS: Nuclei/FG: Connective tissueS: Lignified structures (sclerenchyma, xylem); FG: Non-lignified structures (parenchyma, phloem, collenchyma, epidermis)Identification of lignified vs. non-lignified tissues
Toluidine BlueCationic, MetachromasiaNuclei—Connective tissueLignified structures (sclerenchyma, xylem); Non-lignified (parenchyma, phloem, collenchyma, epidermis)Differentiation of cell wall composition
PASAldehyde group bindingCarbohydratesStarch detectionDetection of polysaccharides
SudanLiposoluble dyeLipids—neutral fatsSuberin detectionLocalization of lipids and suberin
Table 2. Optimized dehydration, clearing, and infiltration protocol for A. thaliana tissues. Modified from [68]. The table summarizes the sequential steps, solvents, and exposure times required for effective paraffin embedding in plant histology.
Table 2. Optimized dehydration, clearing, and infiltration protocol for A. thaliana tissues. Modified from [68]. The table summarizes the sequential steps, solvents, and exposure times required for effective paraffin embedding in plant histology.
StepProcess/SolutionTime of Exposure
1Fixation72 h minimum
2Ethanol 96°2 days
3Ethanol 100°1 day
4Ethanol 100°—Xylol (3:1)2 h
5Ethanol 100°—Xylol (1:1)2 h
6Ethanol 100°—Xylol (1:3)1 h
7Pure XylolOvernight
8Xylol—Paraffin (3:1)2 h
9Xylol—Paraffin (1:1)2 h
10Xylol—Paraffin (1:3)1 h
11Pure ParaffinOvernight
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Valdebenito, S.; Rubio, A.; Moller, A.; Santa Cruz, J.; Castillo, P.; Providell, M.L.; Cáceres, C.; Calbucheo, D.; Hernández, I.; Peñaloza, P. Histological and Immunolabeling Techniques in Arabidopsis thaliana: A Practical Guide and Standardization Roadmap. Agronomy 2025, 15, 2357. https://doi.org/10.3390/agronomy15102357

AMA Style

Valdebenito S, Rubio A, Moller A, Santa Cruz J, Castillo P, Providell ML, Cáceres C, Calbucheo D, Hernández I, Peñaloza P. Histological and Immunolabeling Techniques in Arabidopsis thaliana: A Practical Guide and Standardization Roadmap. Agronomy. 2025; 15(10):2357. https://doi.org/10.3390/agronomy15102357

Chicago/Turabian Style

Valdebenito, Samuel, Alexis Rubio, Alejandra Moller, Javier Santa Cruz, Priscila Castillo, Mayra Lirayén Providell, Camila Cáceres, Diego Calbucheo, Ignacia Hernández, and Patricia Peñaloza. 2025. "Histological and Immunolabeling Techniques in Arabidopsis thaliana: A Practical Guide and Standardization Roadmap" Agronomy 15, no. 10: 2357. https://doi.org/10.3390/agronomy15102357

APA Style

Valdebenito, S., Rubio, A., Moller, A., Santa Cruz, J., Castillo, P., Providell, M. L., Cáceres, C., Calbucheo, D., Hernández, I., & Peñaloza, P. (2025). Histological and Immunolabeling Techniques in Arabidopsis thaliana: A Practical Guide and Standardization Roadmap. Agronomy, 15(10), 2357. https://doi.org/10.3390/agronomy15102357

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