Tissue fixation – the most underestimated methodical feature of

immunohistochemistry

Wilfried Meyer* and Isabelle Nina Hornickel

Institute for Anatomy, University of Veterinary Medicine Hannover Foundation, Germany

*Correspondence to: Prof. Dr. Wilfried Meyer, Institute for Anatomy, University of Veterinary Medicine Hannover

Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany. E-mail: wilfried.meyer@tiho-hannover.de

The present review supplies for the first time a comparative approach referring to negative or positive effects of the most

important and approved fixation media used in immunohistochemistry (Formalin, Bouin`s solution), that are discussed

related to specific impacts caused by the new HOPE® fixation technique. Moreover, possible other influences on the

effectiveness of the fixatives (specimen collection and post-harvesting delay, chemical characteristics of fixatives, fixation

temperature and duration, tissue and organ structure, localization problems of reaction products, approved fixation media

versus HOPE® fixation) are summarised that may have any remarkable consequences for the structural quality and the

immunoreactivities of the different tissues or organs studied. Additionally, information from recent immunohistochemical

findings of the authors was introduced to include personal practical experience of many years.

Keywords: fixation media; chemical characteristics; fixation effects; tissue and reaction qualities; specimen collection;

immunohistochemistry

Introduction

Fixation modifies the physicochemical state, including redox and membrane potentials, of the tissue, and thereby it

changes the reactivity of cellular components with the stain. Consequently, the results of various histological and

histochemical staining methods are modified depending on the prefixation used [1]. In addition, many other parameters

influence the quality and reliability of immunoreactivities, such as the thickness of histological sections, the dilution

range of the antisera used as first layers, and the type or composition of the buffers used for dilution of antisera and of

the chromogens (e.g., DAB or FITC), or as the rinsing solution [2]. However, a critical comparison of different fixation

media is still missing, although bad fixation quality generally strongly impairs the exact localisation of reaction

products in the tissues and organs investigated. An ideal fixation should preserve the original structure of the tissue as

good as possible and should be able to provide an equivalent close to the natural state [3]. This demand can be

accomplished, for example, by fast penetration of the fixation fluid into the tissue, thus avoiding autolysis and

guaranteeing rapid conservation [3 – 5]. In this context, the present review supplies for the first time a comparative

approach referring to the most important fixation media used in immunohistochemistry (IHC), including the new

HOPE® fixation technique. Additionally, information from recent immunohistochemical findings, and from many years

of practical experience of the authors will be introduced [6 – 10] to discuss a relatively broad spectrum of organ systems

studied.

Specimen collection and post-harvesting delay

The preservation of the structural stability of tissues, as closely related to their contents of important substance groups

(proteins, lipids), is one of the most important aspects of histological and histochemical studies. Thus, the first part of

the fixation process must be rapid specimen collection, whereby several factors have to be taken into account to obtain

representative results. It is important to take samples only from freshly dead animals, and to keep the time period

between tissue removal and its immersion in the fixation solution as short as possible!

Our own experience from more than 30 years emphasises a very rapid proceeding to shorten delay between the

availability of the dead animal, sampling and fixation, i.e. the exact time of death of the animal has to be known.

Moreover, the temperature conditions during sampling are of interest because rather warm or cold surroundings

generally accelerate or slow down, respectively, any disintegrative tissue processes [11]. Including the rare information

from the literature, acceptable sampling times for histochemical and, particularly, immunohistochemical purposes

should have delays of not more than 15 - 20 min, as demonstrated for delicate embryonic tissues, whereby about 30 min

post-harvest large vesicles begin to appear, indicating substantial degenerative changes in cell cytoplasm [12]. For adult

tissues, delays should not be longer than 45 – 50 min, such as observed with skin material, e.g. the epidermis [11]. In

the latter case, structural degeneration was first noted in the barrier region between the stratum granulosum and str.

corneum conjunctum. From there such changes spread, independent of storage conditions, from small horizontal

necrotic islands and continuously with increasing pre-sampling times. Storage of the material at 4°C clearly slowed

down this phenomenon but for not more than 4 – 6 hrs [11]. Unavoidable delays between animal death and sampling, as

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possible, for example, when wild mammalian species are concerned, sometimes comprise periods of 6 – 12 hrs, as

known from our own studies on the integument. In these cases we got acceptable fixation for histochemical approaches

only using small specimens after Bouin`s fixation [8], whereas formalin fixation provided only basic structural

information (see also the following paragraphs). However, even when samples fixed in Bouin`s solution, Ca-formol or

the new HOPE® medium are treated optimally in this context, a reduced quality of structure preservation may occur

regarding the latter two fixatives [6]. Nevertheless, several studies have demonstrated that RNA, especially mRNA,

remains more stable in contrast to proteins and can be isolated between 2 - 20 hrs post mortem related to the organ

system in question [for review see, 12].

Last but not least, we have to draw attention to a problem always encountered during the last decades working with

colleagues, doctorands, or PhD students, i.e., the specimens taken often are too large. In our opinion, the specimens

should have a size with an area of not more than 5 x 10 mm and a thickness of not more than 5 mm, so that penetration

with the fixation medium can proceed rapidly to ensure good fixation quality.

Finally, in view of the fact that many modern studies use various types of immunohistochemical and in situ

hybridisation techniques to probe protein and gene expression patterns, it has to be stated again as most important

feature of specimen collection that the time allowed to pass before fixation of the tissue is critical to the results.

Therefore, this time period must be known and strictly controlled so that artifact is not mistaken for a legitimate

staining pattern.

Chemical characteristics of the fixation media

Another general basic factor of a successful fixation process is the composition of the fixation solutions used. To some

extent, the approved and rather reliable Bouin and Ca-formol fixation media are both based on formalin. Formalin

fixation was introduced 1893 by the German medical practitioner Friedrich Blum. He already demonstrated that

formaldehyde forms methylene compounds with amino, amide and hydroxyl groups, thus affecting the solubility and

reactivity of proteins. Formalin belongs to the chemical group of aldehydes, and fixation with these chemicals is a

complex process including a rapid penetration that stops autolysis, followed by covalent binding and cross-linking.

These three parts of fixation proceed simultaneously, but at very different rates (e.g., penetration is 12 times faster than

binding) [3, 13]. It should not be forgotten that formaldehyde causes a decrease in the overall positive electrical charge

of the tissue, which is related to the amount of acid generated during fixation and lowering of the pH. This change of

charge or lowering of the isoelectric point of proteins as a consequence of fixation with aldehydes is bound to alter the

physical nature of proteins with a resulting increase or decrease in the number of sites available for stain uptake [1]. In

contrast to Ca-formol, Bouin`s solution that was introduced 1897 by the French histologist Pol André Bouin,

additionally contains picric acid and glacial acetic acid. This mixture of three ingredients (picric acid by coagulation

and formalin by cross linking representing two different types of protein fixation, acetic acid for penetration

enhancement) has the advantage of penetrating into the tissue more rapidly and evenly, and is less damaging to the

conformation of proteins than formaldehyde alone, thus guaranteeing excellent tissue structure and avoiding tissue

shrinkage already during the fixation step [4, 5, 14]. The good preservation of proteins by Bouin`s solution, in

particular, supplies a sound basis for exact substance localization during immunohistochemical staining experiments [6

– 8].

Bad qualities of structure preservation and immunoreactivity, which sometimes may occur using tissues fixed with

buffered formol, could originate from the high pH level of the solution with 7.0 – 7.4 [for buffer review see e.g., 4],

whereas Bouin`s solution fixes at a pH level of 6.0 to 5.5 (Meyer and Hornickel, personal observations, 2008). Puchtler

and Meloan [15] emphasised that maximum tissue fixation occurs in the pH range 4 to 5.5, no increase in tissue

stabilisation was observed above pH 5.5. They concluded that the increased amount of formaldehyde bound at higher

pH levels only blocks numerous reactive groups. We can support the latter findings and assume that the reduced

antigenicity of the Ca-formol fixed samples is due to the higher pH-level of the latter solution [6, 7].

However, any qualities assumed for the new commercial system, the HOPE® fixation, are difficult to explain as the

selling company offers no detailed description of the ingredients of the HOPE®I solution (also named protection

solution) and the HOPE®II solution. Olert et al. [16], who introduced the HOPE

® fixation into histochemical research,

stated that HOPE®I solution is a hyperosmolar mixture of different amino acids (10 - 100 mmol), with a pH of 5.8 - 6.4,

and works like an immersion fixative. In the second step, the tissue is transferred into a mixture of HOPE® II and

acetone (abs.), followed by an incubation in acetone (abs.). This strong organic solvent acts as a dehydrating agent,

according to the specifications of the manufacturer, and replaces the increasing ethanol concentrations used for the

paraffin embedding of Bouin or Ca-formol fixed tissue. The HOPE® solutions are considered to protect the tissue from

protein cross-linking occurring during dehydration and incubation in the low melting paraffin. From our point of view,

the HOPE® fixation seems to be based mainly on acetone fixation during immersion in the HOPE

® II solution! Acetone

fixates tissue by coagulation of the proteins present. Pure acetone lacks popularity as routine fixative as it does not

preserve tissue structure as good as, for example, formaldehyde [17]. A very negative side effect of using acetone

containing fixation mixtures [e.g., Newcomer`s Carnoy solution, or acetone (abs.) for cryo sections; for other variations

see, 4] is that it can cause strong shrinkage artifacts, as also confirmed by our own observations during many years

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(Meyer, unpublished results, 1970 – 2000). After standard histological staining, HOPE® fixed tissue often likewise

displayed shrinkage artefacts, such as the epithelium rolling up [6]. As acetone is a potent dehydration medium, a solid

crust at the periphery of the tissue might be formed rapidly, causing diminished penetration rates of the paraffin. This

aspect could be an explanation for the lower quality of tissue structure of the HOPE® fixed samples when compared

specifically with Bouin fixed samples [6, 7].

Influence of the temperature and the duration of fixation

An often forgotten but critical aspect is the temperature during fixation. Whereas fixation in Ca-formol and Bouin`s

solution are fairly independent of temperature influences and can be mainly conducted at room temperature [3 – 5], the

first steps of the HOPE® fixation have to be done under constant cooling [16]. In general, low temperatures retard

autolysis [11], but they also decrease the diffusion rate and thus prolong penetration. In our experience, it can be

concluded that it is particularly important to have a temperature of not more than 4°C during the incubation in HOPE®I

and HOPE®II solutions. Even only slightly higher temperatures during this fixation process resulted in a reduced

quality of tissue preservation [6].

Furthermore, the duration of fixation influences fixation quality. Fixing tissue in formalin-based solutions for a time

less than 24 hrs generally results in a mixture of formalin and ethanol fixation. The latter aspect happens during post-

fixative rinsing of the samples in 70% ethanol and during embedding. This means that an interruption of the formalin

fixation before it is completed will lead to cross-linking only at the tissue periphery promoting a crust formation. In

other words, near the centre coagulation occurs, caused by the ascending ethanol solutions during dehydration, or the

centre of the tissue sample remains unfixed including tissue hardening [17].

Moreover, Ca-formol solutions are very susceptible for over fixation problems. Even a controlled prolonged storing

in formaldehyde media may lead to excessive cross-linking and cause irreversible damage of epitopes, which

diminishes the immunoreactivity during IHC experiments [17 – 19]. In the HOPE® fixed tissue, over fixation did not

seem to be much of a problem [6]. This advantage is more relevant regarding Bouin`s fixative that is better suitable for

a longer fixation, because it generally produces no over fixation effects [3; Meyer, unpublished results, 1980 – 2009].

Such quality supports the view of Pol André Bouin, who recommended his solution particularly for embryonic tissues.

Other restrictions of fixation quality may occur realizing that many organs are composed of different tissues types,

which may include varying structural densities with the consequences of varying penetration times of the fixatives

within the organ, producing an only more or less acceptable tissue structure. For example, the esophagus epithelium of

mammals contains great amounts of keratins, in contrast to most of the other organs, and it is surrounded by a rather

voluminous tunica muscularis [20]. Thus, reduced tissue preservation could be a result of such particular cell or organ

stabilizing characteristics. Especially the diminished structural quality of Ca-formol fixed tissue might origin from a

slow penetration rate of the fluid in such structures, in comparison to Bouin`s solution. As already emphasised earlier, a

mixture of different fixatives is still the best way to achieve relatively high quality tissue preservation by a rather steady

progress of solution penetration.

Influences of fixation on immunohistochemical results

According to our experiences, the best preservation of structure as well as continuity of the organ tissue can be obtained

using Bouin`s fixation medium. The HOPE® fixation often showed the next best results, followed by relatively

unsatisfactory results obtained after Ca-formol fixation [6, 7] (Figs. 1, 2). Olert et al. [15] claimed good formalin-like

structure preservation for HOPE® fixed samples. The comparison of results from Hornickel et al. [6] with the findings

of the latter authors emphasises a clearly controversial state on this matter. Unlike the fixation media used in the former

study, common formalin (not buffered formaldehyde in aqueous solution) was applied in the study of Olert et al. [16].

On one hand it can be stated, that tissue preservation of HOPE® is even superior to Ca-formol fixation, but, on the other

hand, it is not as good as Bouin fixation (Fig. 2). Nevertheless, studies conducted on HOPE® fixed human placenta

demonstrated that this new fixation medium can result in a structural quality that is superior to cryo sections [19]. A

comparison of HOPE® and formalin fixation was also subject to other studies, whereby Andrei et al. [21] observed that

structural alterations in the human uterine cervix, such as nuclear distortion and epithelial clefts, were minimal in

HOPE® fixed samples. The findings of a study comparing structure preservation of ovine lung tissue fixed in 10%

neutral buffered formalin or in Bouin`s solution displayed no differences in the quality of structure preservation

between the two fixatives used [22].

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Fig. 1 Demonstration of statistically relevant fixation differences for antibodies in the equine and caprine oesophagus epithelium;

human ß-defensin 2 (hBD2) - dil. prim. antibody: Bouin`s solution and Ca-Formol 1:350, HOPE® 1:1200; Toll-like receptor 2

(TLR2) - dil. prim. antibody: Bouin`s solution and Ca-Formol 1:10, HOPE® 1:120; SB = str. basale, SS = str. spinosum, SS (l) =

lower str. spinosum, SS (u) = upper str. spinosum, SG = str. granulosum; *: p-value < 0.05; after results from Hornickel et al. [6].

Fig. 2 Comparative demonstration of Toll-like receptor 2 in the equine oesophagus epithelium as related to the different fixation

media; dil. prim. antibody: Bouin`s solution and Ca-Formol 1:10, HOPE® 1:120; SB = str. basale, SS = str. spinosum, SG = str.

granulosum; from unpublished results of Hornickel et al. [6].

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In spite of all proven negative aspects of purely formaldehyde based fixation solutions, it is possible to achieve good

immunohistochemical results [see e.g., 10] provided that the following prerequisites are given: a) the specimens have

been collected without any critical time delay, b) the specimens are small or thin and have a homogeneous structure, c)

the buffered formalin solution has been freshly prepared and is changed two to three times during fixation, d) no over

fixation has occurred, e) the dehydration and embedding processes have proceeded correctly, f) the

immunohistochemical procedure has been carried out meticulously, and g) the antibodies available and the visualisation

system are fresh and have been controlled for effectiveness!

One additional astonishing finding of several studies was that the quantities of the primary antibodies could be

reduced using HOPE® fixed tissue, in comparison to formalin-fixed tissues [6, 7, 21, 23]. For example, in most cases of

our study it was possible to reduce the required primary antibody amount usually by 50 - 80%, and for lysozyme even

by 97.5% compared to samples fixed in Bouin`s solution or Ca-formol [6, 7]. Although all authors accentuated such

economical advantage, the question why HOPE® fixation offers this opportunity needs still to be clarified.

Approved fixation media versus modern HOPE® fixation

In contrast to the two well established fixatives Bouin`s solution and Ca-formol, the HOPE® fixation medium is not

formalin based. Therefore it can be concluded that the formation of methylene cross-links [15] is absent in HOPE®

fixed tissue [16]. Nevertheless, the primary characteristic of a fixative is to stabilise tissue structure in order to protect

against shrinking effects during the use of concentrated organic solvents. Olert et al. [16] argue that the direct

application of organic solvents would be an ideal option, as the accumulation of denaturing events that take place during

fixation could be circumvented. This technique is rarely applied due to the incalculable negative affects of organic

solvents on the tissue. Although the exact mechanism of the HOPE® fixation still needs to be elucidated, its aim seems

to be to protect the tissue from the influences of the dehydration solution (acetone) used and to reduce the number of

protein cross-links. For this purpose, the tissue is directly transferred into a special protection solution to weaken the

negative influences of acetone.

Earlier studies already recommended acetone for an optimal retention of antigenic activity in embedded tissue [24].

Nonetheless, the authors emphasised that the major disadvantage of acetone fixation is a considerable hardening and

shrinkage of the material (see beforehand). Such negative side effects should be prevented in the HOPE® fixation

system via previous incubation in a so-called protection solution. As a consequence the reduction of protein-cross links

in HOPE® fixed specimens might directly result in improved preservation of antigenicity. In contrast, formalin fixation

results in a loss or decrease of antigenicity, due to methylene cross-links of reactive sites on proteins making certain

epitopes inaccessible for antibodies. This drawback can be partly overcome by techniques based on heat denaturation or

enzymatic digestion, which facilitate the retrieval of antigens [17, 25]. Some authors argue that this time consuming

procedure could be avoided using the HOPE® fixation as less antigen masking occurs [21, 23]. However, it is important

to stress again that attempts to establish antibodies often remain inconclusive after the use of all three fixatives referred

to beforehand [6]. This finding corresponds with observations of Blaschnitz et al. [19], who argued that inadequate

antibodies lacking specificity cannot lead to satisfying IHC results, and that even the HOPE® technique will not solve

such problems. In conclusion, HOPE® did not result in a direct advantage concerning the establishment of antibodies in

our study [6, 7], which might be related to the quality of the antibodies applied. Nevertheless, supportive evidence was

provided that the improved preservation of antigenicity of HOPE® fixation may be directly related to the effect that the

HOPE® specimens required only 2.5 - 50 % of the amount of primary antibodies in comparison to the other two fixation

solutions.

As discussed by Blaschnitz et al. [19], the main advantage of HOPE® fixation is that it provides the possibility of

applying cryo-compatible antibodies to paraffin sections. It has been demonstrated how antibodies in the human

placenta immunolocalised their antigens on cryo sections and on HOPE® fixed, but not on formalin-fixed paraffin

sections. These results were discussed as related to an improved preservation of soluble proteins in HOPE® fixed

samples, in comparison to samples fixed by the formalin method. The authors highlighted the benefit of the HOPE®

technique for cases when only cryo-compatible antibodies are available. Concerning our study [5, 6], most of the

commercial antibodies had been established for use on paraffin sections. Only the antibody designed for the detection of

mannan-binding lectin (MBL) had been tested on cryo sections and not on paraffin sections. Our results could not

corroborate the findings of Blaschnitz et al. [19], as no positive reactions could be demonstrated on HOPE® fixed

samples for MBL. Nevertheless, the HOPE® fixation can be looked upon as an advantageous tool for fixation when

discussing the aspect of saving time. The HOPE® fixation protocol is shorter than the protocol used for the other two

fixatives, owing to the fact that the dehydration step (increasing ethanol concentrations) and the incubation time in

paraffin is more time-consuming for the Bouin and Ca-formol fixed samples. The dehydration step is limited to the

incubation in acetone for the HOPE® fixed samples.

Finally, in spite of the possible advantages of the HOPE® fixation procedure, it is of great importance to discuss the

HOPE® fixation more critically. One aspect to be kept in mind is the fact the ingredients of the HOPE

® solutions are not

listed or defined in detail by the selling company. It is only known that the solutions contain amino acids [16]. An

interaction of amino acids with antigens is conceivable and can result in a change of epitope structure, which could

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influence the immunoreactivity of the antigens to be detected. A second critical feature is that the tissue sections

prepared should not be stored for more than 7 days (personal communication of the vendor, DCS) before starting the

IHC procedure. During our IHC experiments, we tried to use freshly prepared tissue sections, as false negative reactions

were observed when using tissue sections prepared eight weeks earlier. In contrast, tissue sections of Bouin and Ca-

formol tissue can be used for IHC over years, without any recognisable negative influence on immunoreactivity (Meyer,

unpublished results, 1984 – 2009). A possible explanation for the shorter usability of the HOPE® sections could be the

decreased number of protein cross-links compared to formalin-fixed material (see beforehand), yielding a diminished

stability of proteins and tissue structure. Due to this fact it is most important to keep the sections and paraffin blocks

refrigerated until use after the HOPE® fixation. Another feature somewhat disqualifying HOPE

® from routine

diagnostics is the necessity of refrigerating the samples immersed in the HOPE® solutions. In contrast, there is no need

to refrigerate formalin-fixed samples.

Conclusions

The positive and negative effects of various fixatives on the IHC based detection of antigens have been discussed

extensively in the literature. Independent of our own IHC observations on the esophagus epithelium featuring

particularly the advantages of Bouin`s solution [6, 7], supportive evidence for the fact that fixation in the latter medium

leads to excellent structural and IHC results has been supplied by several authors for different other tissue types. For

example, Smitt et al. [26] obtained the best structurally relevant IHC results for the human cerebellum with Bouin`s

fixative in contrast to formalin. The effect of fixation on ovine lung tissue was studied by Carrasco et al. [25], and they

demonstrated that Bouin`s solution proved to be the most suitable fixative for structural and immunohistochemical

studies. Bedossa et al. [27] investigated the influences of fixation on human liver tissue and also emphasised that

Bouin`s solution was the best fixative for their studies. Other authors discussed the negative effect of formalin fixation

on various tissues [for review see, 4, 20]. For example, Arnold et al. [28] found that neutral buffered formalin was

generally the poorest fixative (in comparison to ethanol and Bouin`s solution) for maintaining antigen recognition by

IHC. Van Alstine et al. [18] discussed the possible effects of over fixation on tissues fixed in formalin in detail. They

concluded that an incubation exceeding three days results in a substantially decreased sensitivity of an

immunohistochemical test. In a more recently conducted study, Buesa [13] summarised the advantages and

disadvantages of formalin fixation. On the one hand the author argued that formalin fixation has been well established

for more than 100 years and is rapid as well as economic. Furthermore, he stated that most antibodies are optimised for

use on formalin-fixed paraffin embedded tissue and that cross-links of proteins are reversible. On the other hand, he

emphasised the carcinogenic potential of formalin. From our point of view this fact is one of the only really important

advantages of the HOPE® fixation method, because its media contain no hazardous ingredients.

We can conclude that the HOPE® fixation technique offers some advantages, especially concerning the absence of

carcinogenic formaldehyde, the cost of antibodies and its short time procedure. If the HOPE® protocol is correctly

established in a laboratory, the preservation of structure is to some extend comparable to samples fixed in Bouin`s

solution. This means that for diagnostic purposes reliable negative or positive results may be obtained, although it is

impossible to achieve differentiated structural information or distribution recognition of reactive substances!

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