Reflections on the Principles of Fixation in Histochemistry and Cytochemistry: With a Special Focus on Immunohistochemistry and In Situ Hybridization

Abstract

In the field of histochemistry and cytochemistry (histocytochemistry), fixation is a critical process for preserving biological structures and enabling accurate analysis. Fixation methods, broadly categorized into precipitating and cross-linking techniques, stabilize biomolecules such as proteins, sugars (carbohydrates) and nucleic acids, although lipids often require specific handling due to the loss during a routine procedure. Traditional staining methods have evolved into advanced techniques like immunohistochemistry (IHC) and in situ hybridization (ISH), which allow for precise analysis of the expression of specific molecules. IHC employs antibodies to visualize specific antigens, with fixation playing a vital role in maintaining antigen integrity. However, excessive fixation can mask epitopes, requiring antigen retrieval techniques to restore antigenicity. Microwave-induced retrieval, for instance, enhances staining efficacy while introducing further fixation by promoting molecular interactions. ISH, which targets nucleic acids with specific base sequences, is also sensitive to fixation conditions. Formaldehyde-based fixatives react variably with purines and pyrimidines, affecting hybridization efficiency with a probe. Positive controls like 28S rRNA help to standardize ISH across facilities, ensuring reproducible and reliable results. Variability in fixation protocols among institutions brings fatal defects in achieving consistent results. Shared standards or the use of robust controls can alleviate these issues, enhancing the accuracy and reliability of histocytochemical analyses for both research and clinical applications.

I. Introduction

In the field of life sciences today, there is a growing need to understand the physiological and pathological states of cells, the fundamental units of life, and to develop methods for their deliberate manipulation. The cell, being too small to be seen, requires innovative and multifaceted approaches for its analysis. Historically, significant information has been accumulated through the microscopic observation of individual cells, with common histological stainings and histocytochemistry. Particularly, in the proper application of histocytochemistry, the process of “fixation” is a fundamental and indispensable step.

Figure 1 depicts an electron micrograph of hepatocytes isolated from rat liver using collagenase perfusion [1]. This work vividly illustrates structures such as glycogen granules, mitochondria, and Golgi apparatus, indicating a highly “resting state.” However, this morphology undergoes substantial changes in response to cellular activities such as proliferation, differentiation, and cell death like apoptosis. Thus, structure and function are intricately linked, making morphological analysis synonymous with functional analysis. To accurately interpret morphology, one must, as Leonardo da Vinci stated, possess the skill of “Super Vedere,” or the ability to see comprehensively. This requires a combination of appropriate methodologies, knowledge, and wisdom. In modern terms, when a cell changes its shape, it is crucial to describe how its biomolecules—proteins, lipids, and carbohydrates—are materially altered. Reviewing the flow of genetic information, DNA is transcribed into mRNA, which is subsequently translated into proteins. Proteins, endowed with enzymatic activities, regulate the metabolism of other molecules such as proteins, sugars, lipids, and nucleic acids. Therefore, proteins should be the primary targets for analysis, followed by an investigation of the components of the central dogma pathway as regulatory mechanisms of their expression [2, 3].

Fig. 1.

Electron micrograph of isolated rat hepatocytes by collagenase perfusion. Hepatocytes were isolated from an adult male rat by collagenase perfusion. The cells were fixed with 2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) (SPB) and post-fixed with 1% osmium tetroxide in 0.1 M SPB, embedded in Epon 812 resin, cut into ultrathin section and observed by a JEOL 100-C electron microscope. For details, please see Reference [1]. Original magnification; 2,900 x.

Historically, histocytochemistry has evolved through various methodologies. Traditional staining methods, such as hematoxylin-eosin, azan, and Giemsa’s staining, relied on properties like electric charge and periodic structure. More advanced techniques like Feulgen staining for DNA and PAS staining for sugars have been developed to detect specific compounds. Furthermore, recent advances have introduced techniques such as enzyme histochemistry, immunohistochemistry (IHC), lectin histochemistry, and in situ hybridization (ISH), enabling the identification of specific proteins, sugars and nucleotide sequences, and now epigenetic factors such as DNA methylation can be analyzed by HELMET [4] and ICON histochemistry [5].

It is important to note two critical points: (1) living cells cannot be “stained”, and (2) life is maintained by the dynamic equilibrium of various biomolecules. Analyzing specific molecular behavior in this equilibrium state is extremely challenging, necessitating the “arrest” of movement—this is where “fixation” becomes essential.

II. What Is Fixation?

Fixation refers to the process of denaturing biological substances, particularly biomolecules such as proteins, sugars, and nucleic acids, to render them insoluble in water. Lipids, however, are generally excluded from fixation because their presence can reduce the transparency during observation. Consequently, a process called “delipidation” is employed to remove lipids, which in other words would not be a direct target of fixation. Nevertheless, fixation of lipids is possible if specific methods targeting lipids are designed, as conventional protocols are insufficient for this purpose.

III. Types of Fixations (Table 1)

Table 1. .

Principles of fixation

Fixation	Remarks	Examples
Precipitating
Dryness	Dehydration	Starch paste, glue
Heating	Heat denaturation	Cooking (boiling, grilling)
Acid	Low pH	Acetic acid, tannic acid
Organic solvents	Low dielectric constant	Acetone, ethanol
Chemicals	Schiff’s reaction	Formaldehyde
Metals	Coordination (single)	Osmium tetroxide
Cross-linking
Chemicals	Between amino residues	Formaldehyde, glutaraldehyde
	Between aldehyde residues	PLP fixative
Metals	Coordination (multiple)	Osmium tetroxide

Fixation methods can be broadly categorized into two types based on their principles: precipitating fixation and cross-linking fixation.

Precipitating fixations

Dry fixation: The simplest method involves drying the sample, causing denaturation and coagulation. This principle is also observed in daily life, such as in the adhesive properties of starch paste or cooked glue.

Heat fixation: Coagulation through thermal denaturation. This is akin to cooking processes like boiling or grilling shrimp and crabs. Upon death, the muscle of these organisms begins to degrade rapidly due to the high reactivity of SH proteases. Heating denatures these enzymes as well as muscle components, preventing further tissue degradation.

Acid fixation: Lowering the pH with acids alters the charge environment, leading to denaturation and coagulation. For instance, soaking fish in vinegar results in a transformation from transparency to opaque white, caused by acid-induced protein denaturation.

Organic solvent fixation: Immersion in organic solvents such as ethanol or acetone reduces the dielectric constant, altering the strength of interaction between ions and precipitating denatured macromolecules. This principle is employed in ethanol precipitation of sugars and nucleic acids, as well as alcohol-based fixation in histocytochemistry or ethanol-based preservation methods in culinary practices. Thus, the principles of fixation are widely applicable, even in everyday life and cooking.

Chemical agents and metallic substances: For example, aldehydes, particularly formaldehyde, are classified as cross-linking fixatives. However, their initial reaction is not truly cross-linking. Fixation begins when carbonyl compounds are formed between the amino groups of proteins and aldehydes, leading to insolubilization. Similarly, molecules coordinated by osmium lose their hydrophilicity and precipitate. Fundamentally, both processes can be regarded as examples of precipitating fixation.

Cross-linking fixations

Examples of cross-linking fixatives include aldehydes such as formaldehyde and glutaraldehyde. Formaldehyde principally reacts with amino groups on proteins to form carbonyl compounds, initiating fixation through insolubilization. Subsequently, the reaction progresses to the formation of methylene bridges through methylol, creating stable cross-links. For sugars, periodic acid oxidation converts adjacent hydroxyl moieties to aldehydes by breaking-HCOH-HCOH-, which then cross-link via amino groups of lysine residues in a process facilitated by agents like periodate lysine paraformaldehyde (PLP) [6]. Osmium tetroxide can also form cross-links by coordinating with multiple molecules.

IV. Remarks on the Choice of Fixatives

As mentioned above, there are various fixation methods available, and the choice of fixative is critically important. Notably, empirical evidence highlights that the initial fixation step has a decisive impact on the outcome. Once a specimen is fixed, the process is basically irreversible, and the effects of fixation, whether significant or subtle, persist throughout subsequent analyses. Incorrectly performed fixation can lead to significant demerits in subsequent investigations. An intriguing analogy can be drawn from culinary practices; drying squid results in “Surume”, boiling or grilling yields different flavors and textures, while pickling in vinegar turns the squid white. Similarly, alcohol immersion results in a unique dish called “Okizuke.” Just as different cooking methods yield different flavors and textures because of changes in ingredients and structures, the choice of fixation method determines the quality of molecular analysis. Selecting the correct fixative ensures optimal visualization and analysis of the interested target molecules.

V. Chemical Natures of Formaldehyde (Table 2)

Table 2. .

Special remarks on formaldehyde

・Formalin is about 40% saturated solution of formaldehyde.

・Formaldehydes are polymerized to paraformaldehyde (PFA). PFA is hydrolyzed to formaldehydes.

・Formaldehydes are changed to methanol and formic acid through the Cannizzaro reaction.

・Amino acid is insolubilized by forming carbonyl compounds with formaldehyde.

・Formaldehyde forms cross-linking between amino residues through methylene bridge.

・Cross-linking formation is promoted under low pH and high temperature.

・Formaldehyde is never stable and easily oxidized under the atmosphere.

・Formic acid formed by oxidation of formaldehyde degrades DNA to apurinic acid.

・Formaldehyde reacts with RNA, but not double-stranded DNA in vivo.

・Formaldehyde reacts with adenine, guanine and cytosine, but not with thymine and uracil due to a lack of exocyclic amino residue.

Considering that formaldehyde is widely used in morphological sciences, especially in pathology, it would be meaningful to refer to formaldehyde in more detail. The fresh and pure formaldehyde solution can be obtained easily by hydrolysis of paraformaldehyde (PFA). Formaldehyde is also commercially available as a nearly saturated solution (~40%), often referred to as formalin. Formaldehyde is known to undergo the Cannizzaro reaction, in which two molecules of formaldehyde participate in a redox reaction to produce one molecule of methanol and one molecule of formic acid. This reaction is particularly pronounced under alkaline conditions. Furthermore, formaldehyde can also autonomously convert into formic acid, through photooxidation or oxidation by atmospheric oxygen. As stated previously, formaldehyde initially reacts with amino groups to form carbonyl compounds. Then, they undergo further reaction resulting in the formation of methylene cross-links between amino groups. These cross-links are highly stable. Although techniques like antigen retrieval claim to “break” these cross-links, the evidence for this is unclear. Cross-links formed between thiol (SH) groups can be disrupted; however, breaking amino group cross-links requires extreme conditions, such as boiling in hydrochloric acid. Special attention should be given to high-temperature and low-pH environments, which accelerate cross-linking reactions. For instance, if the fixation with relatively “old” formalin occurs in a hot room exposed to intense summer heat, self-generated formic acid may lower the pH, promoting excessive cross-linking. This can result in “over-fixation”, which creates significant problems for histocytochemical analysis. The following discussion will examine the impact of fixation on IHC and ISH.

It is also important to note that commercially available formalin typically contains a significant amount of methanol (15–20%) to stabilize it. When using commercial formalin, the process should technically be referred to as “methanol-formalin fixation” rather than simply “formalin fixation”. Methanol is believed to enhance the permeability of formalin, which may make it suitable for standard applications. However, it is essential to remain aware of the chemical influence of methanol in such fixations.

VI. Fixation and IHC

IHC is a technique used to visualize specific antigens, such as proteins, within tissue samples. The two primary methods employed are fluorescence antibody techniques and enzyme antibody techniques. The principle of the latter is illustrated as follows (Fig. 2); antibodies that specifically bind to the target antigen in the tissue are applied. In the enzyme antibody method, the antibodies are conjugated to enzymes such as horseradish peroxidase (HRP). The enzymatic activity is then utilized to generate a visible signal, such as the deposition of an insoluble brown precipitate of DAB oxidation products around the antibody, enabling visualization under a microscope.

Fig. 2.

Principle of enzyme-immunohistochemistry. The principles of direct and indirect methods were shown. In the direct method primary antibody was labeled with horseradish peroxidase (HRP) and in the indirect method secondary antibody was with HRP. As a chromogen solution, 3,3'-diaminobenzidine/4 HCl (DAB) and H₂O₂ were used. (Courtesy of Dr. Paul K Nakane).

It is important to note that antigens in IHC are not limited to proteins; sugars and nucleic acids can also serve as antigens. Additionally, antibodies may recognize specific protein structures. Due to the diversity of antigens involved, IHC is a highly complex technique. To implement IHC effectively, careful consideration of factors related to both the antigen and the antibody is required. On the antigen side, fixation is a critical factor because it can chemically or physically alter the antigen’s epitopes. Fixation may also mask epitopes, rendering them inaccessible, even if the antigen itself remains unaltered. Furthermore, improper fixation conditions can hinder antibody penetration into cells or tissues. On the antibody side, considerations include the antibody’s affinity for the antigen epitope and its overall reactivity (avidity) with the antigen. Chemical changes induced by fixation around the antigen can significantly affect antibody reactivity. For example, even in IgG antibodies, the isoelectric point (pI) varies widely among clones, ranging from 6.4 to 9.0. This variability in pI influences background staining, with alkaline-pI antibodies behaving differently from acidic-pI antibodies. When using polyclonal antibodies in IHC, the mixture of IgG fractions with different pIs can result in variable reactivity depending on the antigen and the surrounding tissue conditions.

VII. Ensuring Reproducibility in IHC

To ensure reproducibility in IHC, it is essential to follow a well-defined protocol. This process involves first determining the fixation conditions, then the conditions for antibody reactions, and finally the conditions for color development. Only by sticking to such a standardized approach can reproducibility be achieved. To assess whether reproducibility has been achieved or to identify potential fluctuant issues, control experiments are indispensable. A common major cause of failure often lies in the fixation process. As generally accepted, one solution to address fixation-related problems is antigen retrieval [7–9]. This approach operates on the principle that strong fixation can be applied initially to keep antigens maximally, and then antigenicity can be restored afterward.

Here, we present our experimental results (Table 3). Mouse intestine was used in this study, where the tissue was fixed with 4% PFA in PBS (pH 7.4) and embedded in paraffin. Immunohistochemistry was performed for histone H3 trimethylated at the fourth lysine residue (H3K4me3), with antigen retrieval conducted using an autoclave (AC). Two buffer conditions, pH 6 and pH 8, were applied during the retrieval process. When signal intensity was evaluated qualitatively using a plus-minus scale, most of the cells, including Paneth cells, revealed similar overall patterns, except the villus epithelium. In this region, the effectiveness of AC varied, indicating that even within the same tissue, the degree of antigen retrieval can differ depending on the cell type. In other words, antigen retrieval does not always occur uniformly across different cell types under the identical fixation and retrieval conditions.

Table 3. .

Effects of antigen retrieval on immunohistochemical detection of histone H3K4me3 in mouse small intestine

Cell types	no AC	AC at pH 8.0	AC at pH 6.0
Paneth cells	+	++ - +++	++ - +++
Epithelial cells
Crypt	+	+++	+++
Villous (basal)	++	++	++
Villous (tip)	+	+	+++

Mouse small intestine (ICR, male) was fixed with 4% PFA in PBS (pH 7.4) and embedded in paraffin. The sections were used for immunohistochemistry for histone H3K4me3 with or without autoclaving (AC) in 10 mM Tris-HCl buffer (pH 8.0) or 10 mM citrate buffer (pH 6.0) [10]. (Unpublished data).

An intriguing property to consider when performing antigen retrieval with microwaving is presented in this slide (Fig. 3). The sample used here is the oviduct tissue of a rhesus monkey. Following ovariectomy (spay), a model of the menstrual cycle was created by administering estrogen (E2) for two weeks, followed by estrogen and progesterone (E2 + P) for an additional two weeks. Frozen sections of the oviducts were prepared, fixed with Zamboni’s fixative—a mixture of neutral picric acid and PFA—and subjected to immunohistochemistry for estrogen receptor (ER)α. In the left column of images, E2 treatment after ovariectomy highlights ERα signals in the nuclei of secretory cells within the oviduct epithelium, while ciliated cell nuclei were negative. When progesterone was added (E2 + P), ERα staining disappeared almost entirely. These results were established with conventional immunohistochemical methods at the time [11]. However, when the tissues and their frozen sections were irradiated with microwave before Zamboni’s fixative, the staining pattern was dramatically changed as shown in the right column; most of all epithelial cell nuclei were now positive in spay and E2 + P specimens while no change was found in E2-treated specimens [11].

Fig. 3.

Effect of microwave (MW) treatment on ERα immunostaining in spayed, E2-treated and E2 + P-treated monkey oviductal fimbriae. Fresh frozen sections of monkey oviducts were treated with (b, d, f) or without MW (a, c, e), and then fixed with Zamboni’s fixative. Then, the sections were reacted with H222 antibody for immunohistochemistry for ERα. In the non-MW-treated tissues from spayed animals, the ER staining was evident in stromal cells (S), but barely detectable in the epithelium (E), whereas MW treatment caused striking increase in the epithelial ER staining. In the E2 primed animals (c, d), ER staining was intensified by MW treatment. In the E2 + P treated animals, ER staining was almost absent in non-MW-treated tissues (e), whereas MW stabilization greatly increased ER staining in both epithelial and stromal cells (f). For details, please see Reference [11]. Original magnification; 400 x.

To confirm which immunohistochemical results are right, we conducted ISH for ERα mRNA. Consequently, ERα mRNA was expressed in almost all epithelial cells in spay and E2 + P oviducts [12], strongly demonstrating the rightness of the sections with microwaving.

What is crucial to note here is that microwaves not only enhance antigen retrieval through superheating effects but also promote fixation by facilitating bonding between closely situated molecules. Understanding this dual property is essential when utilizing microwaves, as both the antigen retrieval and fixation effects should be carefully considered.

VIII. Fixation and ISH

Now, let’s move on to the topic of ISH. ISH is a histochemical methodology used to detect specific base sequences (DNA or RNA) within cell samples or on tissue sections. ISH differs significantly from IHC in a key aspect; its detection target is exclusively nucleic acids (DNA or RNA). The nucleotide bases that make up nucleic acids are classified into purine bases and pyrimidine bases. There are two types of purine bases: adenine (A) and guanine (G). Both contain exocyclic amino groups (amino groups as side chains), which are reactive to fixation methods such as formaldehyde. In contrast, among the pyrimidine bases, cytosine (C) has an exocyclic amino group, whereas uracil (U) and thymine (T) do not. This implies that the regions rich in pyrimidine bases may exhibit reduced fixation efficiency with formaldehyde.

DNA consists of a double-stranded structure formed by complementary base pairs, adenine-thymine (A-T) and guanine-cytosine (G-C). These base pairs are connected by two or three hydrogen bonds, respectively. Due to these hydrogen bonds, the amino groups of these bases do not react with aldehydes. Consequently, double-stranded DNA cannot be fixed directly by formaldehyde. In fact, chemical analysis of in vivo formalin-fixed DNA revealed no formaldehyde reaction products in the DNA molecules [13, 14]. In contrast, RNA, being primarily single-stranded, allows the amino groups of its nucleotide bases to react with formaldehyde. However, it should be noted that during hybridization, the reactive sites with formaldehyde may cause mismatches. Therefore, special care must be taken when designing probes, such as oligo-DNA probes, for targeting mRNA. In our hands, when mRNA is the target, we select regions rich in pyrimidine bases as detection sites. Such regions are less affected by formaldehyde fixation, allowing for more reliable signal detection. This intentional design may minimize the impact of fixation, optimizing the performance of the probes.

The principle of ISH using an oligo-DNA probe is shown in Figure 4. A complementary oligo-DNA sequence is synthesized to match the target mRNA sequence, and a hapten (an antigen molecule) is attached to one or both ends of the probe. This probe is hybridized in situ with the target mRNA, and the resulting signal is ultimately detected using enzyme-immunohistochemistry.

Fig. 4.

Principle of nonradioactive in situ hybridization with oligo-DNA probe. Target mRNA is hybridized in situ with synthetic oligo-DNA probe with complementary base sequences to the mRNA, which was labeled with a hapten such as thymine-thymine (T-T) dimer [15] and digoxigenin (Dig) [16]. Then the section is reacted with anti-hapten antibody and the signal is visualized immunohistochemically. (Courtesy of Dr. Yoshitaka Hishikawa).

In Figure 5, the entire procedure is outlined in a step-by-step format. As for fixation, precipitating fixatives such as ethanol are considered most suitable for DNA. For RNA, however, formaldehyde fixation is likely the best choice. In the pretreatment step, proteinase K is used to expose mRNA, and optimizing these conditions is probably the most challenging aspect of the process. However, once the optimal conditions are established, obtaining reliable results with ISH becomes relatively straightforward [15].

Fig. 5.

Flow chart of non-radioactive in situ hybridization. See the text and for more details, please refer to Reference [15].

Here, we present an example of investigating the effects of fixation on ISH targeting mRNA (Table 4). In this experiment, fresh frozen sections of monkey uterus were fixed with either acetone or paraformaldehyde, and ERα mRNA was detected, as described previously [16]. Generally, the expression patterns of ERα mRNA in the endometrial glandular epithelium and the myometrium were largely consistent. However, when the tissue was fixed with 4% PFA in PBS (pH 7.4), replaced with 30% sucrose, and then used as frozen sections, ERα mRNA expression in the myometrium was completely undetectable. These pre-fixed frozen sections are usually considered suitable samples for ISH due to their excellent preservation of mRNA and hybridization reactions. Upon investigation, it was hypothesized that the low probe permeability in the myometrium is due to actin filaments being arranged in layers, with RNA embedded within them. To address this issue, the use of the surfactant SDS proved effective [17]. This treatment not only improves probe penetration but also imparts a negative charge to the tissue, thereby reducing background signals. However, it presents a drawback of deteriorating tissue morphology. Achieving optimal results requires fine-tuning various conditions for each specific sample through repeated trial and error. This iterative approach is ultimately the most efficient way to obtain reliable and satisfactory outcomes.

Table 4. .

Effects of various fixatives on in situ detection of ERα mRNA in frozen sections of monkey uterus

Fixative	ERα mRNA signal
	Glandular epithelial cells	Myometrium
Fresh frozen
4% PFA (4°C, 10 min)	+	+
4% PFA (RT, 10 min)	++	++
4% PFA (RT, 20–30 min)	+++	+++
Acetone (−80°C, 4 days)	−	−
Acetone (−80°C, 1 day)	+++	+++
Ethanol/acetic acid (3:1)	+	+
Pe-fixed frozen
4% PFA (4°C, 3–4 hrs)	+++	−

Fresh frozen sections of monkey uterus were fixed in 4% PFA in PBS (pH 7.4), acetone or ethanol/acetic acid. In the pre-fixed frozen sections, the uterus was fixed in 4% PFA in PBS (pH 7.4), treated with 30% (w/v) sucrose and then frozen, according to our previous paper [16]. RT; room temperature. Rating scale: −, negative; +, weak; ++, definite; +++, intense.

Here, we should address key considerations regarding formalin fixation, particularly when targeting nucleic acids. As previously mentioned, formaldehyde undergoes spontaneous conversion into methanol and formic acid. Additionally, formaldehyde can be oxidized by exposure to air, further producing formic acid. When DNA is exposed to formic acid, DNA undergoes depurination, leading to the formation of apurinic acid and the degradation of DNA. After the removal of purine bases, the sugar moiety of the DNA retains an aldehyde group. This aldehyde group forms the basis of Feulgen reaction, a well-established method for DNA detection. In the usual Feulgen reaction, DNA hydrolysis to generate aldehyde group is facilitated by heating the sample to approximately 50°C in 0.2 N hydrochloric acid.

IX. Tissue-Related Parameters to be Considered for Successful ISH and Standardization of ISH Signals for Specific mRNA

Having briefly discussed the conditions for hybridization probes in ISH, we now turn to the tissue-related parameters that require consideration. Among these, fixation is the most critical factor. The type and intensity of fixation significantly influence the outcomes of ISH. Key parameters to be considered for in situ detection of specific mRNA include the following:

1. Preservation and hybridizability of mRNA

The integrity of RNA, including its preservation and hybridization capability, is crucial for successful results. RNA is highly susceptible to degradation by RNases, requiring careful handling. Additionally, fixation can chemically modify tissue RNA molecules, potentially hindering hybridization.

2. Accessibility of the probe

The probe must be able to reach the target mRNA. Excessive fixation can impair tissue and intracellular permeability, limiting the probe’s accessibility to its target.

3. Morphological preservation of tissue

ISH involves pretreatment steps and hybridization reactions that can induce various kinds of morphological damage. Therefore, fixation is essential to preserve tissue morphology while maintaining its scientific integrity.

In ISH, since the target is exclusively nucleic acids, it is relatively straightforward to optimize the protocol universally. This is primarily because highly versatile positive controls can be utilized. In our case, we use rRNA as the positive control. rRNA is initially synthesized as 47S pre-rRNA, a big precursor molecule of rRNA which is subsequently processed into 45S pre-rRNA. Through cleavage by rRNA’s intrinsic ribozyme activity, three types of rRNA—18S, 5.8S, and 28S—are produced in a 1:1:1 molar ratio. rRNA constitutes approximately 90% or more of the total cellular RNA, making it an excellent choice for a positive control. We selected 34-base sequences from 18S rRNA and 28S rRNA and designed oligo-DNA probes targeting those regions, as shown in Figure 6.

Fig. 6.

Probe base sequences complementary to 18S and 28S rRNA. 34-base sequences, which are conserved beyond species, were selected from rat 18S [18] and 28S [19] rRNA. The complementary oligo-DNAs were synthesized and used as positive controls of ISH for specific mRNA.

The selected sequence for 28S rRNA is particularly interesting—one might even call it a “miracle.” This is because, at the time [20], this sequence was found to be 100% conserved across all known mammalian species. Moreover, this conservation extends beyond mammals, showing 100% identity in birds, reptiles, and amphibians. Even in lung fish, the sequence remains fully conserved, and in species such as carp and goldfish, there are only minor mismatches. This extraordinary level of conservation is truly surprising. In contrast, no such universally conserved sequence exists in 18S rRNA. For 18S rRNA, we selected a well-conserved sequence within mammals.

Here, we present the results of in situ detection of 28S rRNA (Fig. 7). This experiment was conducted on paraffin sections of PFA-fixed rat liver. Signals for 28S rRNA (right panel) were observed as black staining localized in the cytoplasm and nucleoli. When stained with methyl green and pyronin Y (left panel), the results seemed to be consistent with these findings; nuclei appeared blue due to DNA staining by methyl green, while RNA was stained a brilliant pink by pyronin Y, confirming the reliability of the observed ISH results.

Fig. 7.

Assessment of RNA retention in rat liver sections. Sections of 4% PFA/PBS fixed, paraffin-embedded liver from adult male Wistar rat were stained with methyl green-pyronin Y (MPY) (left panel) and by in situ hybridization for 28S rRNA (right panel). For more details, please refer to Reference [20]. Bar = 50 μm.

Next, we applied this method to human clinical specimens (colon tissue) [20] (Fig. 8). Formalin-fixed paraffin-embedded colon sections were treated with various concentrations of proteinase K at 37°C for 15 min, followed by detection of 28S rRNA (right column). Under the same conditions, methyl green and pyronin Y staining were also performed, but no changes in both staining intensities were observed across the different treatment conditions. In contrast, when detecting 28S rRNA via ISH, an increase in proteinase K concentration resulted in a corresponding increase in hybridization signals. This finding indicated that optimal conditions require higher concentrations of proteinase K; in practice, we used proteinase K at 200 μg/ml in the pretreatment [21]. This positive control system can be applied to other samples as well.

Fig. 8.

Effects of increasing concentrations of proteinase K upon the signal intensities of methyl green-pyronin Y (MPY) staining and 28S rRNA in paraffin sections of human colon. Paraffin sections of formalin fixed human colon tissues were stained with MPY (left column) and by in situ hybridization for 28S rRNA (right column) after pretreatment with increasing concentrations of proteinase K at 37°C for 15 min. MPY staining was not changed irrespective of proteinase concentrations, whereas the signal of 28S rRNA was increased by increasing concentrations of proteinase K, indicating that total RNA did not always reflect hybridizable RNA. For more details, please see the text and Reference [20, 21]. Original magnification; 700 x.

For example, when detecting ERα mRNA in paraffin-embedded rat uterine sections, the optimal proteinase K concentration for 28S rRNA detection was determined to be 10 μg/ml. Under the same condition, ERα mRNA was also efficiently detected (Fig. 9). Furthermore, in the case of clinical specimens where fixation and processing conditions cannot be fully optimized, the presence of a positive control enables calibration of the detected levels of target mRNA expression. This allows for more accurate comparisons of gene expression among different patient specimens. As shown in Figure 10, the expression levels of IL-6 mRNA appear similar between Case 1 and Case 2, but the staining intensity of 28S rRNA is markedly different. By quantifying the 28S rRNA levels in both cases and normalizing the IL-6 mRNA signal intensity to ensure consistent 28S rRNA levels, it was found that IL-6 mRNA expression is significantly higher in Case 1 [22].

Fig. 9.

Effects of increasing concentrations of proteinase K upon the signal intensities of 28S rRNA and ERα mRNA in paraffin sections of rat uterus. Paraffin sections of 4% PFA/PBS fixed rat uterus were hybridized in situ for the detection of 28S rRNA (left column) and ERα mRNA (right column) after pretreatment with increasing concentrations of proteinase K at 37°C for 15 min. The best signal of 28S rRNA was found at 10 μg/ml of proteinase K. And at the same concentration the best signal of ERα mRNA was obtained. For more details, please see the text and Reference [20]. Original magnification; 700 x.

Fig. 10.

Calibration of IL-6 mRNA expression based on 28S rRNA staining. Renal tissues from IgA nephropathy patients with different prognosis, poor (case 1) and better (case 2) were analyzed for IL-6 mRNA and 28S rRNA expressions by in situ hybridization (upper panels). Moreover, both signal densities were measured per unit area of glomerulus by an image analyzer. As shown in the lower panel, IL-6 mRNA signals between both patients were nearly the same, but there was a significant difference in the signals of 28S rRNA. When the signal density of IL6 mRNA was calibrated to that of 28S rRNA, the relative expression of IL-6 mRNA in case 1 was higher than that of case 2, indicating that this approach may be useful especially in the comparative analysis of clinical and pathological specimens. For more details, please refer to Reference [22].

X. Conclusion

As discussed above, the practical concerns regarding fixation is the difficulty of maintaining consistent fixation conditions, not only between specimens but especially across different facilities. In the procedures such as IHC used for companion diagnostics, achieving stable and reliable results becomes challenging when fixation conditions vary among institutions. Therefore, the common use of standardized samples across different facilities could effectively reduce the operational variability, including fixation effects. Ideally, of course, if all facilities could use identical fixation equipment under uniform conditions, this issue would be resolved; however, such consistency is often difficult to achieve in practice. On the other hand, in ISH, it is not always necessary to share standardized samples between facilities. By establishing positive controls such as rRNA, it becomes possible to standardize experimental conditions and calibrate gene expression levels. Beyond fixation conditions, implementing strategies to minimize the impact of human error is essential for improving the precision and reliability of histocytochemical research.