ARTICLE |
Correspondence to: Hidetoshi Ino, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail: ino@med.m.chiba-u.ac.jp
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Summary |
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Antigen retrieval (AR) is frequently required for successful immunohistochemistry (IHC) in archival formalin-fixed, paraffin-embedded tissue sections. Although AR by heating is most generally used, the majority of existing methods are useful only for paraffin-embedded sections. This article describes a simple alternative method for AR that can be used for aldehyde-fixed frozen sections. After fixation in paraformaldehyde, tissue blocks were heated in retrieval solutions and then frozen with dry ice. The optimal temperatures for heating were 90C and above, and the optimal retrieval solutions were distilled water and 10 mM sodium citrate, pH 6.0. Sections were cut with a cryostat and mounted on poly-L-lysine-coated glass slides. After the sections dried, routine IHC was performed. Alternatively, free-floating sections were used. This method not only greatly enhanced the immunoreactivity for a wide range of antigens, especially for nuclear proteins, but also effectively lowered the background staining in some cases. I examined the staining of 14 antibodies using sections of mouse brain and rat testis. The heating process was essential for five antibodies, improved immunoreactivity for seven antibodies, and provided no change for two antibodies. (J Histochem Cytochem 51:9951003, 2003)
Key Words:
antigen retrieval, immunohistochemistry, frozen section, CDK5, ROR, PCNA, cyclin-dependent kinase
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Introduction |
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IMMUNOHISTOCHEMISTRY (IHC) and immunocytochemistry (ICC) are indispensable techniques that provide important information on the localization of proteins in tissues and cells. However, one is not always successful in IHC staining, even if specific antibodies are available and antigens exist in tissues. In many cases, antigen retrieval (AR) is effective for successful immunostaining (
Although AR by heating is widely effective, the majority of the existing methods are designed for paraffin-embedded sections. Frozen sections have been generally exempt from these methods because such sections are fragile and are easily destroyed by heating. The author reports here an alternative AR method by heating, which is useful for paraformaldehyde-fixed frozen sections. The optimal conditions, including the retrieval solution, temperature, and time, were examined. This method is quite simple but effective and needs no special equipment except for a cryostat. We have already reported the successful IHC detection of several proteins using this method (
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Materials and Methods |
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Preparation of Tissue Sections
Male and female ddY mice (body weight 3040 g) and male SpragueDawley rats (body weight 250300 g) were perfused via the heart with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.5 (80 ml, 5 ml/min for mice; 400 ml, 25 ml/min for rats). Mouse brains were coronally or parasagittally sliced at 35-mm thickness. Rat testes were scissored in the tunica albuginea. The tissues were further fixed in the same solution at 4C overnight. The testes were cut in half. The tissues were then transferred to retrieval solutions and incubated at 4C overnight. The tissue blocks were placed in a small metal basket and immersed in 300 ml of preheated retrieval solutions with gentle stirring using a hot plate for 3 min or 10 min. The heating temperatures tested were 50C, 60C, 70C, 80C, 90C, or boiling (9799C). This heating step was omitted for the control tissue blocks. Then the blocks were immediately placed in cold 30% sucrose in PBS and incubated at 4C overnight. The heat-treated and untreated (control) blocks were immersed in an embedding medium (Tissue-Tek OCT Compound; Sakura Fine Technical, Tokyo, Japan), quickly frozen with crushed dry ice, sectioned with a cryostat at 10-µm thickness, and mounted on glass slides coated with freshly prepared 0.02% poly-L-lysine. The sections were dried well for more than 1 day in a vacuum desiccator at room temperature (RT). The frozen tissue blocks could be stored at -80C for several months and the sections could be stored in a desiccated chamber at -20C for several months. To prepare free-floating sections, the frozen tissue blocks were cut with a cryostat at 40-µm thickness and the sections were stored in PBS containing 0.2% sodium azide at 4C.
All animals were treated and cared for in accordance with the Chiba University School of Medicine guidelines pertaining to the treatment of experimental animals.
Immunohistochemistry
Sections were reacted on glass slides (slide-mounted sections) or in wells (free-floating sections). The sections were immersed in 0.3% Triton X-100 in PBS for 3 hr at RT and then in a blocking solution (5% skim milk in PBS) at 4C for 46 hr. After brief washing with PBS, the sections were reacted with primary antibodies diluted with the blocking solution at RT overnight. The primary antibodies were as follows: rabbit polyclonals, anti-cdk5 (C-8; Santa Cruz Biotechnology, Santa Cruz, CA; 1:2000), anti-c-fos (Ab-5; Oncogene Research Products, Cambridge, MA; 1:5000), anti-Egr-1 (C-19; Santa Cruz; 1:2000), and anti-cdk2 (M2; Santa Cruz; 1:2000); goat polyclonals, anti-ROR1 (C-16; Santa Cruz; 1:2000), anti-Fos B (102; Santa Cruz; 1:2000); and mouse monoclonals, anti-PCNA (19F4; Boehringer Mannheim, Mannheim, Germany; 1:2000), anti-PCNA (PC10; Pharmingen, San Diego, CA; 1:5000), anti-cdc2 p34 (17; Santa Cruz; 1:2000), anti-tyrosine hydroxylase (MAB318; Chemicon International, Temecula, CA; 1:2000), anti-neuronal nuclei (NeuN; Chemicon; 1:2000), anti-GFAP (G5A; Boehringer Mannheim; 1:200), and anti-vimentin (V9; Boehringer Mannheim; 1:200). After washes with PBS (8 min, four times), the sections were reacted with a secondary antibody, biotinylated anti-rabbit, goat or mouse IgG(H+L) antibody (Vector Laboratories, Burlingame, CA; 1:200) at RT for 3 hr. After washes with PBS, the sections were reacted with the reagents of the Vectastain ABC Kit (Vector) at RT for 1 hr. After subsequent washes, the sections were stained with 0.3 mg/ml 3,3'-diaminobenzidine, 0.6 mg/ml ammonium nickel (II) sulfate hexahydrate, and 0.0009% hydrogen peroxide in 50 mM Tris-HCl, pH 7.5.
Brief Protocol for Antigen Retrieval
For convenience, the brief protocol for AR is listed here. Optimal conditions for immunostaining should be tested in each case.
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Results |
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To demonstrate the effect of AR by heating, first the author compared immunostaining on slide-mounted sections, those that were boiled for 3 min in distilled water and others that were not (untreated). Then the conditions for AR were examined, including the retrieval solution, temperature, and heating time. Finally, the effect of AR on immunostaining by the free-floating method is shown. In the following experiments, sections were stained using the avidinbiotin system with 3,3'-diaminobenzidine, but the same retrieval effect was obtained with fluorophore-conjugated secondary antibodies (data not shown). In the absence of primary antibodies, no staining was observed in heat-treated tissues (data not shown).
CDK5 and ROR in the Mouse Brain
The remarkable effect of the AR method on the immunostaining of two neuronal proteins, CDK5 and ROR, in the mouse cerebellum is shown. CDK5 is one of the cyclin-dependent kinases enriched in the nervous system. CDK5 is located in both the nuclei and cytoplasm of neurons (
is an orphan nuclear receptor strongly expressed in the cerebellum and thalamus (
was occasionally observed in untreated sections, the signal was weak and unstable (Fig 1C). With heating, clear nuclear staining of ROR
in Purkinje cells and molecular layer cells was demonstrated (Fig 1D).
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NeuN, GFAP, and Tyrosine Hydroxylase (TH) in the Mouse Brain
NeuN and GFAP are frequently used as cell markers for neurons and astrocytes, respectively. NeuN is a neuron-specific transcription factor located in both the nuclei and cytoplasm, and GFAP is an intermediate filament protein located in the cytoplasm. TH, a key enzyme mediating catecholamine metabolism, is located in the cytoplasm of catecholaminergic neurons. Although NeuN, GFAP, and TH were observed in untreated sections (Fig 2A, Fig 2C, and Fig 2E), heating enhanced their immunoreactivity (Fig 2B, Fig 2D, and Fig F). The AR effect on GFAP and TH immunoreactivity was conspicuous. More clearly stained GFAP-positive astrocytes and TH-positive dopaminergic neurons were seen in heat-treated sections than in untreated sections. In addition, heating lowered the background staining in these three cases.
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c-Fos, FosB, and Egr1 in the Mouse Brain
c-Fos, FosB, and Egr1 are transcription factors called immediate-early genes because of their immediate induction after several stimuli. c-Fos is widely used as a marker of neuronal activity. Although these proteins are greatly induced by stimuli, certain levels of these proteins exist in the normal mouse brain. All were detected in untreated sections. Although c-Fos and FosB immunoreactivities were slightly enhanced by heat treatment, the effect was not dramatic (data not shown). Staining of Egr1 was almost uninfluenced by heating (data not shown). Staining of these proteins was seen in the nuclei.
PCNA, CDC2, CDK2, CDK4, and Vimentin in the Rat Testis
The effect of AR on immunostaining of these proteins in the rat testis was tested. PCNA is a protein necessary for DNA synthesis. It has been reported that, for detection of PCNA, AR by heating is effective in paraffin-embedded sections (
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Examination of Retrieval Conditions
Antigen retrieval by boiling tissue blocks in distilled water for 3 min was effective for a number of antibodies. The author further examined the heating conditions, including the retrieval solution, temperature, and heating time.
First, the composition and pH of the retrieval solutions were assessed under the fixed conditions of boiling for 3 min (Table 1). Citrate and bicarbonate buffers were chosen because these buffers were used occasionally in AR by heating with paraffin-embedded sections. For most of the antibodies, 10 mM sodium citrate, pH 6.0, was as effective as distilled water as a retrieval solution. Although in the cases of CDK5 and CDC2, 100 mM sodium citrate, pH 6.0, and 10 mM sodium bicarbonate, pH 9.0, were less effective, the immunoreactivity of the other antibodies showed equivalent levels of enhancement in these buffers. In contrast, 10 mM sodium citrate, pH 3.0, was not suitable as a retrieval solution, except in the cases of GFAP and TH. Sections heated in this buffer showed uneven staining (the peripheral area of the sections was not stained although the central area was strongly stained).
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Next, the heating temperature and time were assessed using 10 mM sodium citrate, pH 6.0 (Table 2). Intense nuclear staining of CDK5 was observed at temperatures of 70C and above. For PCNA immunostaining with PC10, heating at 90C and above for 3 min or at 80C for 10 min was necessary. For 19F4, heating at 90C for 10 min or boiling for 3 min was needed. For demonstration of nuclear staining of CDC2, tissues should be heated at 90C for 10 min or boiled for 3 min.
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In addition, the background-lowering effect of heating, which was observed in the cases of NeuN, GFAP, and TH, was seen at temperatures of 90C and above.
Effects of AR on Free-Floating Sections
Immunostaining by the free-floating method is often used for brain sections. The effect of AR by heating on free-floating sections was examined for CDK5 and ROR (Table 3). Nuclear staining of CDK5 was not observed in untreated slide-mounted sections, as mentioned above. However, weak nuclear staining was occasionally found in untreated free-floating sections. With heat treatment either in distilled water or 10 mM sodium citrate, pH 6.0, strong enhancement of immunoreactivity was observed. In the same way, the weak nuclear immunoreactivity of ROR
in free-floating sections was greatly enhanced by heat treatment.
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Blocking of Endogenous Pseudoperoxidase Activity by Heating
Antigen retrieval by heating is effective for IHC with several antibodies, as described. In addition, there is one secondary beneficial effect. Endogenous pseudoperoxidase activity mainly derived from erythrocytes was completely blocked by boiling. Therefore, special treatment for blocking endogenous pseudoperoxidase was unnecessary.
Morphological Changes of Tissues Caused by Heating
With heating, the surface of the tissue blocks underwent artifactual morphological changes, such as vacuole formation and irregular arrangement of cells (data not shown). However, the interior portions retained their normal morphology, similar to unheated tissues (compare brain tissues in Fig 1 and Fig 2 and seminiferous tubules in Fig 3). In general, the damage caused by heating in 10 mM sodium citrate, pH 6.0 was less serious than that in distilled water. Tissues heated in a low ionic strength solution swell, possibly as a result of the imbalance in osmotic pressure, and this may be one of the factors responsible for the tissue damage.
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Discussion |
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Preparation and rehydration of paraffin-embedded sections are sometimes laborious and time-consuming if there is no equipment such as an automated paraffin processor. The method described here enables AR by heating for cryostat sections. Advantages of the method are as follows: (a) fixed frozen sections that are slide-mounted and dried can be used; (b) no special equipment is necessary; and (c) the effect of AR is remarkable for a wide range of antibodies.
As described above, this method showed extreme effects on CDK5, ROR, PCNA, and CDC2 immunostaining, and was effective for NeuN, GFAP, TH, c-Fos, FosB, CDK2, and vimentin immunostaining. There was no remarkable effect on CDK4 and Egr1 immunostaining. Antigen retrieval methods on free-floating sections, in which tissues were heated before slicing as done here, have been reported (
For AR by heating in paraffin-embedded sections, factors influencing retrieval efficiency, including pH, molarity, calcium ion concentration, and chemical composition of the retrieval solution, have been examined. It has been reported that the pH is more critical than the chemical composition of retrieval solutions (
Heat-treated slide-mounted sections can be used for in situ hybridization and TUNEL, the technique most frequently used to detect apoptotic cells in tissue sections. Enhancement of the TUNEL reaction by microwave irradiation on paraffin-embedded sections has been reported (
In spite of the many advantages, there are some disadvantages to this method: (a) heat treatment damages tissues and (b) the heating effect is not always evenly distributed in tissues. Damage caused by heating is serious in soft tissues. However, many paraformaldehyde-fixed tissues, including the brain, spinal cord, lung, liver, thymus, spleen, kidney, alimentary tract, testis, and ovary of mice and rats, are viable after heating. Mouse and rat fetuses, at least after embryonal day 14, can be used. The heating temperature should be lowered or the heating time should be shortened for soft tissues. However, at present this method cannot be used for cultured cells. In addition, the unevenness of the heating effect in tissues is inevitable. Although the surface of the tissue is seriously damaged, its interior structure is retained. Damaged surface sections should be abandoned and undamaged sections should be collected. The AR effect on large tissues may be insufficient in the most interior part. It would be necessary to cut tissues to an appropriate size. Although there was no apparent problem in the present study using paraformaldehyde-fixed mouse brains and rat testes, the relationship of the AR effect to the size and sort of tissues and fixation conditions should be examined using larger tissues.
In conclusion, the antigen retrieval method reported here is simple and requires no special equipment. This method is useful for researchers who prefer frozen sections.
Received for publication August 28, 2002; accepted March 19, 2003.
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