Heat-Induced Antigen Retrieval for Immunohistochemical Reactions in Routinely Processed Paraffin Sections

Abstract

The development of heat-induced antigen (epitope) retrieval (HIER) technologies has led to dramatic improvements in our ability to detect antigens in formalin fixed, archival tissues. Paradoxically, wet heat treatment at temperatures greater than 95°C in appropriate buffer solutions can reconstitute the antigenicity of many proteins that have been rendered nonreactive during the fixation and paraffin embedding process, which heretofore could only be identified in fresh or frozen tissues. The reason for this effect is unclear, but it has been suggested that the vigorous heat treatment partially reverses or disrupts the aldehyde cross-links occurring in proteins during formalin fixation and restores the original conformation of antigenic epitopes. The great success of antigen/epitope retrieval technologies further emphasizes the importance of preanalytical steps in immunohistochemistry. Over the past several years, since this technology was first reported, there have been numerous modifications to the original formulation. It is the purpose of this chapter to discuss the critical issues required for optimal HIER and to provide guidelines for the use of popular HIER buffers and heating devices used for routine immunohistochemical detection.

1. Introduction

Immunohistochemistry has become an essential adjunct of modern diagnostic pathology. However, successful staining is highly dependent on the preparation of the tissues prior to immunohistochemical staining. Traditionally, most pathological tissue samples are fixed in formaldehyde and embedded in paraffin wax in preparation for examination of microscopic morphology. Unfortunately, many potentially interesting antigens become altered during tissue fixation and processing, interfering with their immunohistochemical detection. To overcome this obstacle, snap-frozen tissue sections had been a preferred alternative to formalin-fixed paraffin-embedded tissue sections. However, the morphology of frozen section immunohistochemistry is relatively poor, the use of frozen tissue necessitates additional equipment for freezing and storing frozen tissues, and the pathologist must decide in advance which tissues to freeze for immunohistochemical studies. By contrast, paraffin sections offer well-preserved tissue architecture and cytomorphology, are routinely prepared and readily available in every pathology department, and require no special handling or additional equipment. Furthermore, formalin-fixed paraffin-embedded tissues represent an invaluable source of human tissues for retrospective immunohistochemical studies (1). As a result of these considerations, pathologists and other investigators have continually sought methods for recovering the antigenicity of fixed tissues.

Alterations of antigens in paraffin-embedded tissues are related to a variety of changes in the three-dimensional structure (conformation) of proteins due to cross-linking by formaldehyde and to a lesser extent to heating and dehydration during paraffin embedding (2). As a consequence, the antigenic determinants (epitopes) are destroyed, denatured, or masked, which may diminish or abrogate their detection. The earliest attempts to retrieve antigenicity utilized proteolytic enzymes (3, 4), which presumably act by breaking formaldehyde-induced methylene cross-links in the antigenic molecules. Although proteolytic methods have been especially useful for retrieval of cytokeratins, they are difficult to control, and careful attention is needed to optimize their retrieval effect and avoid tissue destruction. Other approaches using coagulative agents such as heavy metals, alcohols or acid solutions with or without formaldehyde for tissue fixation, and pretreatments with cold 20% sucrose-saline or with sodium methoxide solutions have also been tested to improve immunostaining of specific antigens (5, 6).

1.1. Wet Heat-Induced Epitope Retrieval

Despite some successes with the above pretreatments, the development of wet heat-induced epitope retrieval (HIER) procedures, which involves heating the fixed tissue sections in dilute metal-salt or buffer solutions at or above 100°C, for several minutes to 1/2 h, was the critical breakthrough in paraffin section immunohistochemistry (2, 7-9). Today, there are many variations of the original HIER technique. These differ primarily in the recommended buffer solutions and/or the source or mode of heating, but the basic formula of wet heat treatment over a fixed time period is similar. The most popular HIER technologies use microwave ovens, stainless steel or plastic pressure cookers, autoclaves, vegetable steamers or water-baths as the heat sources and low molarity buffers with acidic or alkaline pH (8, 9, 11-14).

HIER methods have dramatically expanded the universe of antibodies that react in formalin-fixed paraffin-embedded tissues. Antibodies that never before reacted in paraffin section show specific staining following HIER pretreatment. Moreover, HIER substantially increases the sensitivity of reactions of the majority of antibodies directed to formaldehyde-resistant antigens as well. Additionally, appropriate HIER minimizes the problem of over-fixation, reducing the immunostaining differences found between the 24-h fixed material and tissues that have been kept in formalin for days or even weeks (see Note 1). HIER technology has also enabled immunohistochemists to routinely stain a wide spectrum of antigens in etched, epoxy resin-embedded sections for bone marrow diagnosis (10).

The exact mechanism by which HIER works is unknown. Hydrolytic cleavage of formaldehyde-related chemical groups and cross-links, unfolding of inner epitopes, and the extraction of calcium ions from coordination complexes with proteins are among the hypothesized mechanisms (15-17). Antigens that are hidden in 4% formaldehyde-fixed tissues may become available without HIER, if a coagulative agent such us acetic acid or mercuric chloride is added to the formaldehyde or coagulative agents are used for fixation without formaldehyde (see Fig.1). Coagulative agents reduce the cross-linking potential of formaldehyde, suggesting the possible involvement of formaldehyde cross-links in the antigen masking effect (2). The involvement of formaldehyde cross-links in antigen masking is also supported by the observation that tissues fixed in coagulative or mixed (formaldehyde and coagulant) fixatives need substantially shorter HIER times for antigen recovery than for tissues fixed in 4% formaldehyde alone. On the other hand, as a result of its cross-linking ability, formaldehyde fixation alone provides better antigen definition and morphological preservation after HIER than does mixed or neat coagulative fixatives (see Fig.1).

Fig.1.

Detection of CD3 T-lymphocyte antigen in 16-h formalin-fixed, paraffin-embedded reactive human tonsil sections. In the absence of HIER the antigen is not detectable in the tissue fixed in neat formaldehyde, but it is stained in tissues fixed either in mixed (formaldehyde and coagulant) or in coagulative fixatives. Increasing the HIER treatment time (PC 2.5′) does not substantially improve immunostaining, but adversely affects tissue morphology.

In addition to ongoing efforts to refine pathologic diagnoses, the rapid development of molecularly targeted therapies and the need to identify these targets or their surrogate markers in pathologic tissues have continued to drive the demand for improvements in antigen identification and the development of new antibodies. New generations of highly specific antibodies directed against peptide sequences of lymphocyte subset antigens (e.g. CD4, CD10, CD79a), oncoproteins (i.e. bcl-2, cyclin D1, p53), or molecules of prognostic and/or predictive relevance in cancer (i.e. CD117, Her2/c-erbB2, Her1/EGFR, estrogen and progesterone receptors) that require or benefit from HIER have been introduced, and their number is expanding rapidly. As a consequence, immunophenotyping of paraffin-embedded tissues, coupled with an appropriate HIER technique, has attained a central role in the modern immunohistochemistry laboratory.

In this chapter, we briefly summarize the current principles of HIER and offer protocols for its reliable use in paraffin-embedded tissues. The broad methodological repertoire now available provides great latitude for laboratories using HIER, but also underscores the need for standardization to achieve better intra and interlaboratory reproducibility. There are several quality assurance organizations that offer proficiency testing programs and provide valuable technical information on their websites designed to promote standardization (see Note 2).

1.2. Factors that Influence HIER

1.2.1. Composition of Retrieval Buffer

Experimental data suggest that the amount of heat and the duration of heating followed by the pH and chemical composition of the retrieval buffers are the most important factors for efficacy of HIER (7, 8, 15-18). Citrate buffer (sodium citrate–citric acid) at pH 6.0 is a very popular retrieval medium and has been used at molarities between 0.01 and 0.1 M (7, 8). Detergents (e.g., 0.1% Tween 20) added to the standard citrate buffer may improve the performance (14), but more prominent tissue deterioration and foaming may also be experienced. Other widely used antigen retrieval buffers are Tris-HCl at various concentrations (0.1–0.5 M) at alkaline pH (8.0–10.0) (7, 11, 14, 17, 19), EDTA-NaOH (0.1 M) at pH 8.0, and the mixture of 0.1 M Tris and 0.01 M EDTA at pH 9.0 (15, 16, 19). The EDTA-containing solutions provide excellent antigen recovery, but treated tissues may show enhanced tissue damage as compared to citrate-based retrieval buffers. Other HIER solutions have also been tested, but are not as widely used as those described above (7, 8).

There are a number of companies that offer prepared “proprietary” antigen retrieval buffers. Of the commercially available citrate-based antigen retrieval buffers, we have used the pH 6.0 Target Retrieval Solution (TRS) from Dako (Carpinteria, CA) with excellent results (20, 21). TRS can be used to retrieve epitopes that are not otherwise detectable in formalin-fixed, paraffin-wax sections (i.e., CD5/Leu1; CD35/To5), and it can substitute for enzymatic digestion in the detection of other antigens, e.g. CD21/1F8; CD35/Ber-MAC-DRC and BerEP4 (20, 22) (see Fig.2).

Fig.2.

Detection of antigens in 16-h formalin-fixed, paraffin-embedded reactive human tonsil. (a) Demonstrates retrieval of Ki67 and bcl2 antigenicity following HIER treatment using standard citrate buffer and a commercial citrate-based buffer (TRS; DakoCytomation). The TRS buffer shows slightly better retrieval. (b) Poor retrieval for CD35 using standard citrate buffer and excellent retrieval using the TRS buffer. Trypsin (0.1%) shows intermediate level of antigen retrieval. Use of “higher efficiency” buffers Tris pH 8.0 and Tris–EDTA pH 9.0 did not provide significant retrieval (not shown).

1.2.2. Heating Devices

A wide variety of heating devices have been adapted for use in HIER, including microwave ovens (MWO), pressure cookers, vegetable steamers, autoclaves, and water-baths. The most reproducible results may be achieved with MWOs that incorporate time and temperature control even above the atmospheric boiling point (combined with a plastic pressure cooker). The archetypes are the professional laboratory microwave instruments, which allow the full control of time/temperature/power and vacuum (see Note 3). Owing to of the high cost of these instruments, most immunohistochemistry laboratories utilize simpler consumer type microwave ovens. The most important factors to be considered in choosing a commercial MWO are the presence of (1) a digital timer for precise time adjustment and (2) a turntable for uniform heating of the retrieval solution. MWOs can either be used at their maximum power of 700–1,200 W, or at a reduced power of 250–300 W. The advantage of using reduced power is that heavy boiling and evaporation can be avoided, while maintaining the buffer temperature near 100°C. However, the duration of treatment should be extended by ~40% of the corresponding treatment time at maximum power. MWO-mediated HIER can also be performed in plastic pressure cookers designed for household kitchen applications (see Subheading 3.2), which appear to eliminate some of the drawbacks (e.g., heavy boiling and evaporation) of the standard MWO protocols. Metallic tools should never be used in the MWO, therefore, heat-resistant plastic Coplin jars or containers and heat-resistant plastic slide holders with a capacity of 15 sections and above are required for this application.

The use of stainless steel kitchen pressure cookers (PC) is another simple instrument for achieving reproducible HIER (see Subheading 3.4) (see Note 4). The heating device is usually an inexpensive commercial electric hotplate with at least 1 kW of power. Careful use of a PC provides uniform heating without heavy boiling and allows one to treat large batches of slides (9, 13, 19). The PC method eliminates the need for careful monitoring during the retrieval that is necessary in full-power MWO method to prevent accidental drying out of the sections as a result of evaporation. Recently, time and temperature controlled electric heating devices have been commercialized to achieve more standard results, which work either at atmospheric pressure such as the PT (LabVision, Fremont, CA) or under supra-atmospheric pressure such as the Pascal (Dako) and the Decloaking chamber (Biocare Medical, Concord, CA).

Wet (hydrated) autoclave treatment represents a uniform heating method and is preferred over MWO irradiation by some authors (12, 17) (see Subheading 3.5). The main disadvantages of the autoclave method are availability and prolonged procedure time. In an effort to standardize heating conditions of HIER, water-baths have recently been recommended (see Subheading 3.6), e.g. for the FDA-approved Herceptest. However, water-baths are necessarily limited to 100°C. Vegetable steamers (e.g., Model HS900; Black and Decker, Towsen, MD) have also been employed successfully for HIER and represent yet another inexpensive heat source. Like water-baths, their maximum temperature is limited to 100°C.

1.2.3. Temperature and Duration of Treatment

The power of the heating device, the composition, pH, the volume of buffer, and the degree of tissue fixation are the major factors that determine the duration of a particular HIER protocol. The efficacy of antigen retrieval is also influenced by the duration and conditions of cooling following heat treatment (see Subheadings 3.1-3.6) (see Note 5). The recent technique using heated buffer solutions (without organic solvent) for dewaxing and rehydration of sections (e.g. Trilogy; Cell Marque, Hot Springs, AR) will also contribute to the retrieval effect and should be considered when HIER is designed. Automated immunostainers combining dewaxing with additional HIER pretreatment followed by the full immunostaining sequence (e.g., BenchMark; Ventana Medical Systems, Tucson, AZ) provide highly reproducible and standard results.

Most methods employ temperatures near or above 100°C. Heating above the atmospheric boiling temperature is possible in traditional or in microwavable pressure cookers as well as in autoclaves (see Subheading 1.2.2). The best commercial PCs can reach an operating pressure of about 103 kPa/15 psi, resulting in ~120°C temperature (19, 21). It is important to emphasize that the maximal temperature achievable in pressure cookers depends upon the particular model used (see Note 4). When utilizing autoclaves as the heat source, 120°C is easily achievable and is the recommended standard (12, 17, 21).

Under identical heating conditions, i.e. temperature and duration, buffers of alkaline pH (pH 8–10) such as the common Tris or EDTA-containing HIER buffers generally result in more efficient antigen retrieval than the conventional citrate buffer at pH 6.0. The performance of both buffer types can be improved using higher temperatures or extending the time of treatment. However, there is a point at which no significant additional benefit can be achieved and at which continued treatment will begin to result in tissue deterioration. Generally, maximal antigen recovery is reached well before significant tissue damage occurs, but this time point can be influenced by the degree of tissue fixation. As a general rule, the high pH and EDTA-containing buffers reach their optimal treatment time much faster than do the lower pH citrate-containing buffers. It is important to be aware that prolonged treatment with EDTA-containing buffers may attack soda-lime glass below the adhesive layer, facilitating tissue detachment. Table 1 lists some of the popular HIER buffers according to their efficiency/aggressiveness under the same heating conditions. Guidelines on the use of some of these popular HIER buffers and heating devices are summarized in Table 2.

Table 1.

Common antigen retrieval buffers grouped according to their HIER efficiency and aggressiveness on tissue morphology

Group 1 Higher efficiency buffers	– EDTA, pH 8.0 (1–100 mM)
Shorter incubation times	– TRS high, pH 9.9 (DakoCytomation)
Most tissue destructive	–Tris–EDTA, pH 9.0 (10 mM/1 mM)
	–TRIS pH 10.0 (10–200 mM)
Group 2 Lower efficiency buffers	– TRS, pH 6.1 (DakoCytomation)
Longer incubation times	– Boric acid, pH 7.0 (20 mM)
Less tissue destructive	– Citrate, pH 6.0 (10 mM)

Table 2.

Guidelines for the use of HIER buffers and heading devices

Heat source	Buffer type	Buffer volume	Temperature	Time of exposurea,b	Comments
Professional-type MWO with temp. control	Group 1Group 2	1,000 mL	98°C (set)	15–20 min 20–25 min	Total heating time (from room temp.)
Professional-type MWO with temp. control	Group 1Group 2	1,000 mL	120°C (set)	1.5–2 min 2–4 min	Time at working temperature
Consumer-type MWO at full power	Group 1Group 2	90–150 mL	100°C (boiling)	2–3 ×5 min 3M: × 5 min	Total heating time (from room temp.)
Consumer-type MWO at full power 700–1,200 W	Group 1Group 2	600–800 mL	100°C	18–20 min 24–30 min	Total heating time (from room temp.)
Consumer-type MWO at low power 300 W	Group 1Group 2	100–150 mL	100°C	28–30 min 30–35 min	Total heating time (from room temp.)
Traditional PC (5 L)	Group 1Group 2	3,000 or 150 mL in plastic slide container	~120°C	1.5–2 min 2–4 min	Time at full pressure
Microwave PC (NordicWare)	Group 1Group 2	1,500 or 150 mL in plastic slide container	~110°C	25–30 min 30–40 min	Total heating time (from room temp.)
Autoclave	Group 1Group 2	Autoclavable container with buffer	120°C (set)	12–15 min 15–20 min	Total heating time (from room temp.)
Water-bath	Group 1Group 2	Coplin jars with preheated buffer	95–99°C	25–30 min 40–45 min	Time at working temperature

^aThe duration of HIER within the indicated range depends on the duration of formaldehyde fixation

^bExtending HIER beyond the indicated duration, particularly with Group 1 buffers, may be tissue destructive

Firm attachment between the tissue and the glass slide is crucial to prevent detachment of the section during HIER. Mounting the sections on 3-amino-propyltriethoxysilane (APES)-coated glass slides creates suitable adhesion to withstand the HIER procedure. Alternatively, charged glass slides, such as SuperFrost (Ultra) Plus (e.g. Fisher Scientific), provide highly standard adhesion even under extreme HIER conditions. Heat activation of the binding between the adhesive layer and the tissue section by melting the wax, for at least 30 min, is crucial before dewaxing.

If unacceptable tissue deterioration occurs, reducing the duration of heating and/or the subsequent cooling period or decreasing the ionic strength of the retrieval buffer may improve the cytomorphological preservation. In some cases, inferior nuclear morphology, due to partial loss of histone proteins, can be compensated with extended counterstaining. Some antigens may be destroyed or washed out by the higher temperature, i.e. extracellular antigens, glycophorin C, and surface immunoglobulin light chains. Therefore, careful titration of HIER is needed when a new antibody is tested in these applications.

The most rapid HIER procedures utilize the standard MWO or the household pressure cooker, whereas the more time-consuming procedures use the MWO/pressure cooker combination, the autoclave, vegetable steamer, and the water-bath. In the full-power MWO procedures, treatment is performed in 2–4 heating cycles of 5 min each. Each cycle is interrupted to replenish evaporated buffer in the Coplin jars containing the slides. MWO can also be used without disruption either with larger buffer volumes of 600–800 mL at full power or with a Coplin jar volume of buffer but at limited power (~300 W). The duration of treatment should be extended in the latter case. The household pressure cooker procedure may take up to 40 min, while the combined use of MWO and pressure cooker can exceed 60 min (see Subheadings 3.1 and 3.2). “Hot start” modifications of the latter method, in which buffer is preheated before placement of the slides within the pressure cooker (see Subheading 3.3), can shorten the treatment time considerably. Wet autoclave HIER is probably the most time-consuming method, since the total time required from start-up to cool-down of the autoclave might be up to 2 h (see Subheading 3.5) (17). The water-bath method operating just below 100°C may also take 40 min or more (see Subheading 3.6).

2. Materials

1. Tissue sections mounted on adhesive glass slides, e.g. Super Frost (Ultra) Plus (Fisher Scientific) and heat activated at 60°C (3–5-μm thick paraffin sections) or at 85°C (1–2-μm thick epoxy resin sections) for at least 30 min.

2. Standard domestic microwave oven at 700–1,200 W, with a turntable for constant temperature and an electronic timer control.

3. Plastic Coplin jars or other microwaveable slide containers and suitable slide racks.

4. Microwaveable pressure cooker of 3-L volume (MWPC) (Nordic Ware, Minneapolis, MN).

5. Stainless steel commercial pressure cooker of 5.5-L volume (e.g., Prestige Model 6193, Prestige Group UK Plc., Lancashire, UK).

6. Electric hot plate rated at 1,000 W.

7. Laboratory autoclave, e.g. Sanyo MAC 235 EX (Sanyo, Osaka, Japan).

8. Standard laboratory water-bath rated at 100°C.

9. Antigen retrieval buffer solutions (see Table 1).

10. Post-HIER blocking buffer: 0.05 M Tris-buffered saline (TBS pH 7.6) containing 2% low-fat milk powder.

11. Immunostaining buffer: TBS containing 1% BSA or normal goat serum.

12. Test antibodies: mouse monoclonal anti-Ki67/Mib1 (1:150), anti-bcl2/124 (1:100), anti-CD35/To5 (1:100), and rabbit polyclonal anti-CD3 (all from Dako). Incubation: 60 min at room temperature. (For immunostaining protocols, see Chapters 25, 26, 28, and 29)).

13. Antimouse Ig-s polymer-conjugate, EnVision+™ detection system (Dako; K4001). Incubation: 30 min at room temperature.

14. DAB+/H₂O₂ chromogen-substrate kit (Dako; K3468). Incubation: 10 min at room temperature.

3. Methods (See Note 6)

3.1. Standard MWO Antigen Retrieval: Basic Method (7,8,17, 19,21)

1. Transfer the dewaxed or deplasticized (see Note 7) rehydrated and methanolic peroxide blocked slides into plastic Coplin jars or containers filled with a HIER buffer (see Table 1 and Note 8).

2. Fill remaining positions in the Coplin jars or plastic slide racks with blank slides (see Note 9).

3. Place the Coplin jars or plastic containers in the center of the microwave’s turntable, cover containers with loose-fitting lids or screw caps, and heat at maximum power (700–1,200 W). The time of irradiation depends upon the power setting of the microwave, the type of container used, and the volume of buffer (see Table 2). The solution should boil for 3–5 min. A large capacity microwaveable plastic container filled with 600–800 mL of HIER buffer may require a ~30–45 min continuous heating cycle (13).

4. After the heat cycle, add distilled water into the container to replenish any evaporated buffer (see Note 10). Repeat the heat cycle up to three times. Alternatively, uninterrupted heating at low power (250–300 W) for ~30 min may be performed without the risk of unwanted drying out of sections due to vigorous boiling and evaporation.

5. Following the necessary number of heating cycles, allow the slides to cool for approx. 20 min (see Notes 5, 11, and 12).

6. Proceed immediately with the immunostaining protocol (see Notes 13-17 and Chapters 25, 26, 28, and 29).

3.2. Microwave Pressure Cooker Method (14)

1. Place the dewaxed/deplasticized and rehydrated sections (see Notes 7 and 8) in an MWPC filled with 1,500 mL HIER buffer (see Tables 1 and 2 and Note 18).

2. Place the MWPC inside the MWO. Heat at maximum power (700–1,200 W) for 30–40 min with a standard buffer volume and slide number (see Notes 9 and 19). Under these conditions, it will take about 15–20 min to reach maximum temperature. The slides should then remain at maximum temperature for less than 20 min (see Notes 20 and 21).

3. Release the pressure carefully and cool the sections immediately in post-HIER blocking buffer (see Subheading 2, step 10) for 15–20 min before immunostaining (see Note 10). Alternatively, the slides can be allowed to cool down in the open pressure cooker (approx. 20–30 min) and then transferred into the post-HIER buffer (see Notes 5, 11, and 12).

4. Proceed immediately with the immunostaining protocol (see Notes 13-17 and Chapters 25, 26, 28, and 29).

3.3. “Hot Start” Variation for Microwave Pressure Cooker (14)

This variation is performed as described above in Subheading 14.3.2., except that the sections are placed into buffer preheated to about 95°C, and irradiation is performed for only 8–15 min in the closed MWPC. Under these conditions, maximum heat is attained in 3–5 min.

3.4. Standard Pressure Cooker Method (9,13)

1. Preheat 3 L of HIER buffer (see Tables 1 and 2) to a boiling point in a stainless steel 5.5 L capacity pressure cooker (Prestige) without sealing the lid, using an electric hot plate as a heat source. Alternatively, to conserve buffer, the pressure cooker may be filled with distilled water, and a small loosely covered container filled with the selected buffer may be placed into the pressure cooker to hold the slides (see Note 18).

2. Place the dewaxed/deplasticized and rehydrated slides in metal slide racks and immerse in the hot buffer (see Notes 7 and 8). Seal the PC and bring to full pressure, which is attained when both the “rise-n-time” indicator and the safety plug are in the upright position. Treat the sections at full pressure for 2–3 min (see Note 4).

3. Release the pressure and cool the PC under running tap water for approx. 10 min (see Note 5).

4. Transfer slides into post-HIER blocking buffer for 15–20 min (see Notes 10-12) and continue with the immunohistochemical staining procedure (see Notes 13-17 and Chapters 25, 26, 28, and 29).

3.5. Autoclaving (12,21)

1. Place the dewaxed/deplasticised and rehydrated slides into metal or heat-resistant plastic slide racks (see Notes 7 and 8) and immerse in an autoclavable incubation container filled with 250 mL HIER buffer (see Tables 1 and 2). Cover the container with a loose-fitting lid to avoid evaporation.

2. Autoclave at 120°C (2 atm) for a maximum time of 20 min.

3. As soon as the autoclave can be opened, the slides should be washed in the post-HIER blocking buffer (see Notes 10-12) for 15–20 min, then proceed with the immunostaining (see Notes 13-17 and Chapters 25, 26, 28, and 29).

3.6. Water-Bath Heating (21)

1. Immerse the Coplin jar(s) into the water-bath and preheat HIER buffer (see Table 1) to the required temperature (95–99°C).

2. Immerse the dewaxed/deplasticised and rehydrated slides into the preheated HIER buffer and cover the Coplin jar with a loose-fitting lid to avoid evaporation (see Notes 7 and 8).

3. Continue with HIER for the required time, which is usually 40 min (see Table 2).

4. Incubate the slides in the post-HIER blocking buffer for 15–20 min (see Notes 10-12) then proceed with the immunostaining (see Notes 13-17).