Bcl-2 and Bax proteins are present in interphase nuclei of mammalian cells

The Bcl-2 family of proteins comprises both cell death inhibiting and cell death promoting members, generally believed to be cytoplasmic and predominantly membrane-associated. Like Bcl-2, many Bcl-2-related proteins contain a C-terminal membrane insertion domain and much research is aimed at evaluating the functional role of their localization to the outer membranes of mitochondria, the endoplasmic reticulum, and perinuclear membranes. However, confocal fluorescence microscopy of human breast cancer cells and rat colon cancer cells immunostained with commercial antibodies raised against different epitopes of the anti-apoptotic Bcl-2 and the pro-apoptotic Bax protein revealed that these proteins are not only present in the cellular cytoplasm, but also within interphase nuclei. This was confirmed by Western blot analysis of isolated nuclei. In human cells, certain epitopes of Bcl-2, but not of Bax, were also found to be associated with mitotic chromatin. Anti-estrogen treatment of human breast cancer cells or transfection with antisense bcl-2 led to a reduction in both cytoplasmic and nuclear Bcl-2. Transfection of human bcl-2 and bax into rat cells resulted in cytoplasmic and nuclear Bcl-2 and Bax. This data seems in line with increasing evidence that the role of the Bcl-2 family of proteins should be extended to activities inside the nuclear compartment.     

Immunohistochemical detection and distribution of the 1,25-dihydroxyvitamin D3 receptor in rat reproductive tissues

Vitamin D₃, via its active metabolite 1,25-dihydroxyvitamin D₃, plays a critical part in male and female reproduction in the rat. 1,25-Dihydroxyvitamin D₃ activity is mediated by an intracellular receptor (VDR). VDR distribution in reproductive tissue has not been studied using antibodies against the receptor. We developed a polyclonal antibody against the VDR and used it to examine VDR distribution in male and female rat reproductive tissues. In rat testes, VDR epitopes were observed in seminiferous tubules, specifically in spermatogonia, Sertoli cells and spermatocytes. Spermatozoa stained faintly. Epithelial cells of the epididymis, seminal vesicles and prostate also expressed VDR epitopes. In the female rat reproductive tract, immunostaining for VDR was seen in ovarian follicles, specifically in granulosa cells. Weaker VDR immunostaining was observed in follicular thecal cells and in the ovarian stroma and germinal epithelium. Corpus luteal cells stained intensely for VDR. Epithelium of fallopian tubes and the uterus also contained VDR epitopes. Both nuclear and cytoplasmic VDR immunostaining was observed in male and female rat reproductive tissues. We conclude that the VDR is widely distributed in male and female reproductive tissues and that it is likely to mediate actions of 1,25-dihydroxyvitamin D₃ in the tissues.     

Different modes of sialyl-Tn expression during malignant transformation of human colonic mucosa

Monoclonal antibodies TKH2 and B72.3, which react with the mucin-associated sialyl-Tn(STn) antigen, preferentially bind to cancerous but not normal colonic tissues. If O-acetyl groups are removed by saponification of tissues, MAb TKH2 will react with normal colonocytes, whereas MAb B72.3 remains non-reactive. To explain this difference in binding specificity, we tested both MAbs against synthetic constructs of single (monomeric) or clustered (trimeric) STn epitopes by enzyme immunoassay. Both MAb TKH2 and MAb B72.3 reacted with trimeric STn, but MAb TKH2 demonstrated greater binding than MAb B72.3 to monomeric STn. This suggests that normal colonic mucosa expresses monomeric STn epitopes, but that with transformation to malignancy, clustered STn epitopes appear. The appearance of clustered STn epitopes during colonic carcinogenesis represents a novel pattern of carbohydrate antigen expression and implicates alterations at the level of apomucins and/or glycosyltransferases responsible for cluster epitope formation.     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed,paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed, paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

Identification of monoclonal antibodies that recognize novel epitopes in native chondroitin/dermatan sulfate glycosaminoglycan chains: their use in mapping functionally distinct domains of human skin.

Five monoclonal antibodies (MAb), 7D4, 4C3, 6C3, 4D3, and 3C5, were produced in mice immunized with high buoyant density embryonic chick bone marrow proteoglycans (PGs) as antigen. All of these MAb recognized epitopes in native chick bone marrow and cartilage PGs which could be selectively removed by chondroitinase ABC and chondroitinase AC II, indicating that their epitopes were present in chondroitin sulfate glycosaminoglycans (GAGs). These MAb recognized epitopes present in purified cartilage PGs obtained from a wide variety of different vertebrate species. However, none of the new MAb detected epitopes in Swarm rat chondrosarcoma PG. On the basis of these results, we propose that these MAb recognize novel epitopes located in chondroitin sulfate/dermatan sulfate glycosaminoglycan (CS/DS GAG) chains, representing at least four and possibly five different structures. Immunocytochemical studies have shown that the epitopes identified by these new MAb are differentially distributed in tissues. All of these MAb immunocytochemically detected epitopes in embryonic chick cartilage and bone marrow. Three of them (4C3, 7D4, and 6C3) recognized epitopes in adult human skin. All three detected epitopes in the epidermis, one (6C3) strongly detected epitopes in the papillary dermis, and two (4C3, 7D4) detected epitopes in the reticular dermis. Immunostaining patterns in skin using the new MAb directed against native CS/DS structures were distinctly different from those obtained using MAb against the common CS isomers. The distribution of these CS epitopes in functionally distinct domains of different tissues implies that these structures have functional and biological significance.     

Isolation of monoclonal antibodies recognizing rat bone-associated molecules in vitro and in vivo.

Knowledge of the number and kinds of differentiation steps that characterize cells of the osteoblast lineage is inadequate. To further analyze osteoblast differentiation, we generated a series of monoclonal antibodies (MAb) to osteogenic cells. Spleen cells from mice immunized with whole-cell populations enriched for expression of osteoblast-associated properties or bone formation in vitro were fused with the SP2/0 myeloma cell line. Supernatants from growing hybridomas were screened by indirect immunofluorescence on frozen sections of a portion of 21-day fetal rat heads that included the calvaria bone, periosteum, muscle, fibrous connective tissue, and skin. Six MAb were selected with bone-associated staining and limited ability to label other tissues. Either cell surface or cytoplasmic molecules were recognized by five of the MAb; one recognized a molecule detectable both in the cytoplasm, on the cell surface, and in the extracellular matrix. Of the antibodies selected, one identified both preosteoblasts and osteoblasts and has been found to be against alkaline phosphatase. The others recognized the mature osteoblasts, osteocytes, and chondrocytic cells. The pattern and distribution of the labeling in vivo extended to primary cells and cell lines in vivo. These results support earlier observations on molecules differentially expressed by cells at different stages of the osteoblast lineage and extend the available cell surface and cytoplasmic epitopes identifiable as marker molecules.     

Increased Membrane/Nuclear Translocation and Phosphorylation of p90 KD Ribosomal S6 Kinase in the Brain of Hypoxic Preconditioned Mice

Our previous studies have demonstrated that hypoxic precondition (HPC) increased membrane translocation of protein kinase C isoforms and decreased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the brain of mice. The goal of this study was to determine the involvement of p90 KD ribosomal S6 kinase (RSK) in cerebral HPC of mice. Using Western-blot analysis, we found that the levels of membrane/nuclear translocation, but not protein expression of RSK increased significantly in the frontal cortex and hippocampus of HPC mice. In addition, we found that the phosphorylation levels of RSK at the Ser227 site (a PDK1 phosphorylation site), but not at the Thr359/Ser363 sites (ERK1/2 phosphorylated sites) increased significantly in the brain of HPC mice. Similar results were confirmed by an immunostaining study of total RSK and phospho-Ser227 RSK. To further define the cellular populations to express phospho-Ser227 RSK, we found that the expression of phospho-Ser227 RSK co-localized with neurogranin, a neuron-specific marker, in cortex and hippocampus of HPC mice by using double-labeled immunofluorescent staining method. These results suggest that increased RSK membrane/nuclear translocation and PDK1 mediated neuron-specific phosphorylation of RSK at Ser227 might be involved in the development of cerebral HPC of mice.     

Expression of a novel nuclear protein is correlated with neuronal differentiation in vivo

We report the production of a monoclonal antibody (MAb 526) that recognizes a novel, developmentally regulated nuclear protein expressed in neurons throughout the rat nervous system. Analysis of whole brain and cell nuclear extracts by SDS-PAGE and immunoblotting determined that MAb 526 recognizes a single nuclear protein (np) of apparent molecular weight 42 kD, designated np526, as well as a slightly larger (ca. 44 kD) cytoplasmic protein. Light microscopic immunocytochemistry showed np526 to be present in neurons of all types throughout the central and peripheral nervous systems. Nuclei of both fibrous and protoplasmic astrocytes were also immunoreactive, but oligodendrocyte nuclei were negative. Positive, but highly variable immunocytochemical staining of nonneural cell nuclei in a variety of other tissues was also observed. Electron microscopic (EM) immunocytochemistry using pre-embedding peroxidase methods revealed that np526 is associated with euchromatin or with the edges of condensed chromatin bundles in neurons, indicating that it is likely to be a chromosomal protein. Most interestingly, the expression of np526 was found to be developmentally regulated in brain. Immunocytochemical analysis of the developing cerebral cortex from embryonic day (E) 16 to postnatal day (P) 4 and cerebellum from P4 to P18 revealed that np526 first appears in central neurons following the cessation of mitosis and that the intensity of nuclear staining increases during subsequent neuronal maturation. To our knowledge, np526 is the first presumptive chromosomal protein whose expression has been precisely correlated with the early postmitotic differentiation of mammalian neurons.     

Expression of a novel nuclear protein is correlated with neuronal differentiation in vivo.

We report the production of a monoclonal antibody (MAb 526) that recognizes a novel, developmentally regulated nuclear protein expressed in neurons throughout the rat nervous system. Analysis of whole brain and cell nuclear extracts by SDS-PAGE and immunoblotting determined that MAb 526 recognizes a single nuclear protein (np) of apparent molecular weight 42 kD, designated np526, as well as a slightly larger (ca. 44 kD) cytoplasmic protein. Light microscopic immunocytochemistry showed np526 to be present in neurons of all types throughout the central and peripheral nervous systems. Nuclei of both fibrous and protoplasmic astrocytes were also immunoreactive, but oligodendrocyte nuclei were negative. Positive, but highly variable immunocytochemical staining of nonneural cell nuclei in a variety of other tissues was also observed. Electron microscopic (EM) immunocytochemistry using pre-embedding peroxidase methods revealed that np526 is associated with euchromatin or with the edges of condensed chromatin bundles in neurons, indicating that it is likely to be a chromosomal protein. Most interestingly, the expression of np526 was found to be developmentally regulated in brain. Immunocytochemical analysis of the developing cerebral cortex from embryonic day (E) 16 to postnatal day (P) 4 and cerebellum from P4 to P18 revealed that np526 first appears in central neurons following the cessation of mitosis and that the intensity of nuclear staining increases during subsequent neuronal maturation. To our knowledge, np526 is the first presumptive chromosomal protein whose expression has been precisely correlated with the early postmitotic differentiation of mammalian neurons.     

Fibronectin of human liver sinusoids binds hepatitis B virus: identification by an anti-idiotypic antibody bearing the internal image of the pre-S2 domain.

Anti-idiotypic antibodies (anti-Ids) have been successfully used to characterize and isolate receptors of several cell ligands. To prepare an immunological probe for identification of cellular components interacting with the hepatitis B virus (HBV), polyclonal antisera against a panel of five HBV-specific monoclonal antibodies (MAbs) were produced in syngeneic BALB/c mice. MAbs to HBV used for immunization (Ab1) recognized biologically important and potentially neutralizing epitopes, located in the pre-S1, pre-S2, or S region-encoded domains of HBV proteins. All the anti-Ids (Ab2) were specific to idiotopes of the homologous Ab1 and inhibited their interaction with the corresponding viral epitopes, suggesting that they recognized unique determinants on the paratope of each immunizing Ab1. Therefore, all five generated polyclonal anti-Ids were of the Ab2 beta type and could represent internal images of viral epitopes. Ab2 raised against the pre-S2 region-specific MAb F124 bound to the extracellular matrix fibronectin of human liver sinusoids. Immunohistochemical studies demonstrated the attachment of viral and recombinant (S, M) hepatitis B surface antigen particles with the pre-S2 region-encoded epitopes to the fibronectin of human liver sinusoids. In contrast, recombinant (S, L*) hepatitis B surface antigen particles, in which the epitope recognized by F124 MAb was not expressed, did not show any binding capacity. These findings suggest that human liver fibronectin may bind HBV in vivo by the pre-S2 region-encoded epitopes in a species-restricted manner. Furthermore, binding of the circulating virus to liver sinusoids could facilitate its subsequent uptake by hepatocytes.     

A monoclonal antibody to chicken gizzard desmin that recognizes intermediate filaments and nuclear granules in BHK21/C13 cells

One hybridoma (AC54), which produces monoclonal antibody (MAb) that recognizes both intermediate filaments (IFs) and nuclear granules in BHK21/C13 cells, and two hybridomas (AC19 and AC36) which produce MAbs that recognize IFs only, were obtained by using a crude actin preparation from chicken gizzard as an antigen. In immunoblotting, both the AC54 and AC19 MAbs reacted with the 52 kD protein (desmin) and some other proteins in gizzard and BHK21/C13 cells. Indirect immunofluorescent microscopy of BHK21/C13 cells showed that the cytoplasmic filaments stained by these MAbs were IFs based on their colchicine-induced whorl formation. The ability of AC54 MAb to recognize IFs was more limited than that of AC19 MAb. The nuclear granules recognized by AC54 MAb were in a different location than the cytoplasmic IFs and sometimes were concentrated in the nucleolus. These results indicate that AC54 MAb is an anti-desmin MAb that reacts with some desmin-related proteins; that it recognizes IFs differently than AC19 MAb, another anti-desmin MAb; and that it recognizes nuclear granules in locations where desmin or desmin-related protein has not yet been reported.     

Immunonucleochemistry: a new method for in situ detection of antigens in the nucleus of cells in culture

The advancement of immunocytochemistry (ICC) allows one to observe detailed spatial distribution of cellular antigens, but, with some limitations. Using conventional ICC, it is difficult to distinguish the nuclear localization from cytoplasm, as two large subcellular compartments overlap on the z-axis. In this study, we have investigated whether in situ immunostaining of ‘naked’ nuclei could provide an unambiguous method for detection of nuclear antigens. We have designed a protocol that efficiently lyses plasmalemma, while keeping the nuclear envelope intact. The optimal condition for lysing the plasmalemma was 0.5% Nonidet P-40 for 5 min in both neuronal and non-neuronal cultured cells. Using this protocol, we could unambiguously isolate nuclear from cytoplasmic ICC signals. Since the present protocol has been designed for immunostaining of ‘naked’ nuclei from cultured or isolated cells, we have coined a new term to refer to this procedure as ‘immunonucleochemistry’ (‘INC’ for abbreviation).     

Comparative Study of Antigen Retrieval Heating Methods: Microwave, Microwave and Pressure Cooker, Autoclave, and Steamer

We present a study comparing the most popular beating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HC1 buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR immunostaining. All heating methods yielded good results for AR immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies; the microwave pressure cooker, extended microwave heating (5 min × 4) and autoclave heating then showed a similar intensity of staining that was stronger than results obtained with the steamer (20 min) or regular microwave heating (5 min × 2 J. Extension of the steamer heating time, however, yielded equivalent results. This study indicates that different heating methods can yield similar intensities of AR immunostaining if the heating times are adjusted appropriately. It is noteworthy that, in general, the adjusted conditions for maximal retrieval differ from those most widely cited in the literature, or recommended by manufacturers. That several heating devices may provide similar results permits the use of different AR heating methods according to the equipment available. This study also is an early step in standardizing the AR immunostaining protocol by providing uniform conditions for “maximal retrieval” as a common end point for all laboratories.     

The presence of 19-kDa Bcl-2 in dividing cells

The 26-kDa bcl-2 gene product inhibits apoptosis and cell proliferation. Cleavage of Bcl-2 into a 22-kDa fragment inactivates its anti-apoptotic activity and is a key event in apoptosis. Here, and in recent work, we describe massive 19-kDa Bcl-2 immunoreactivity in non-apoptotic cells, suggesting a link with viability rather than cell death. Loss of 19 kDa Bcl-2 in adriamycin-induced apoptotic cells underlines this. G2/M-phase accumulation of cells by nocodazole-treatment also results in loss of 19 kDa Bcl-2. Next to its well-documented cytoplasmic localization, a substantial pool of Bcl-2 resides in nuclei. Hampered nuclear localization of Bcl-2 leads to a loss of cell cycle repression. This has led us to point at a pivotal role for nuclear Bcl-2 in cellular proliferation. In this report, cellular fractionation of bcl-2 transfected cells in various phases of the cell cycle reveals a constitutive cytoplasmic pool of 19 kDa Bcl-2. Nuclear 19-kDa Bcl-2 immunoreactivity is far more pronounced in rapidly dividing nuclei compared with more quiescent nuclear fractions. This implicates that ongoing cell proliferation involves cleavage of nuclear Bcl-2 with a 19-kDa fragment.     

Nuclear localization of the titin Z1Z2Zr domain and role in regulating cell proliferation

Titin (also called connectin) is a major protein in sarcomere assembly as well as providing elastic return of the sarcomere postcontraction in cardiac and striated skeletal muscle tissues. In addition, it has been speculated that titin is associated with nuclear functions, including chromosome and spindle formation, and regulation of muscle gene expression. In the present study, a short isoform of titin was detected in a human osteoblastic cell line, MG-63 cells, by both immunostaining and Western blot analysis. Confocal images of titin staining showed both cytoplasmic and nuclear localization in a punctate pattern. Therefore, we hypothesized that human titin may contain a nuclear localization signal (NLS). A functional NLS, 200-PAKKTKT-206, located in a low-complexity, titin-specific region between Z2 and Z repeats, was found by sequentially deleting segments of the NH₂-terminal sequence in conjunction with an enhanced green fluorescent protein reporter system and confirmed by site-directed mutagenesis. Overexpression of titin's amino terminal fragment (Z1Z2Zr) in human osteoblasts (MG-63) increased cell proliferation by activating the Wnt/β-catenin pathway. RT-PCR screens of tissue panels demonstrated that residues 1–206 were ubiquitously expressed at low levels in all tissues and cell types analyzed. Our data implicate a dual role for titin's amino terminal region, i.e., a novel nuclear function promoting cell division in addition to its known structural role in Z-line assembly. Wnt; catenin; osteoblast     

Mechanisms of heat-induced antigen retrieval: does pH or ionic strength of the solution play a role for refolding antigens?

We investigated the effects of pH and ionic strength of solutions used for antigen retrieval to elucidate the mechanism of heat-induced antigen retrieval (HIAR) in immunohistochemistry. The immunostaining intensity of nuclear, cytoplasmic, cell membrane, and extracellular matrix antigens with 17 different antibodies was evaluated in formaldehyde-fixed and paraffin-embedded mouse and human tissues. Deparaffinized sections were autoclaved for 10 min in buffers with different pH values ranging from 3.0 to 10.5. To test the influence of ionic strength on immunoreactions, the sections were autoclaved for 10 min in 20 mM Tris-HCl buffers (TB) at pH 9.0 and 10.5 with or without 25, 50, and 100 mM NaCl. There were two immunostaining patterns for pH dependency of HIAR. First, the majority of antibodies recovered their antigenicity when heated in the buffers with both acidic pH (pH 3.0) and basic pH (pH 9.0 and 10.5). Second, some antibodies showed strong immunostaining only at basic pH values (pH 9.0 and 10.5). When the sections were autoclaved in TB at pH 9.0, immunostaining of all eight antibodies examined decreased as the NaCl concentration increased. On the other hand, when the sections were treated with TB at pH 10.5, all antibodies yielded stronger reactions in the buffer containing NaCl than in the buffer without NaCl; five antibodies exhibited the strongest immunoreaction at concentrations from 25 to 50 mM. These results suggest that the extended polypeptides by heating are charged negatively or positively at basic or acidic pH, and that an electrostatic repulsion force acts to prevent random entangling of polypeptides caused by hydrophobic attractive force and to expose antigenic determinants, during cooling process of HIAR solution.     

Nuclear and mitotically enhanced epitope.

Salt-extracted proteins of taxol-stabilized microtubules from Chinese hamster ovary cells arrested at mitosis were used to immunize mice for hybridoma production. From a group of related monoclonal antibodies (MAbs), one, C9, recognized an epitope on antigens localized by immunofluorescence microscopy to interphase centrosomes and nuclei. The availability of the nuclear antigen was cell cycle-dependent; however, permeabilization of cells before fixation revealed that the antigen was present throughout the cell cycle. The nuclear antigen was exposed during prophase and was released from the nucleus upon nuclear envelope breakdown filling the cytoplasm of the mitotic cell. Antigenic material re-accumulated at daughter nuclei and was concealed during G1 phase. Detergent extraction of the cytoplasmic antigen from mitotic cells enabled localization of antigens to centrosomes, kinetochores, and the furrowing region/midbody. Immunoblot analysis of cells of a variety of species of origin identified an approximate 250 kD polypeptide as corresponding to the nuclear antigen, whereas polypeptides of 107/117 kD as well as approximately 250 kD accounted for the mitotic cytoplasmic antigens. No polypeptides could be associated with antigens at centrosomes, kinetochores, or midbodies. This MAb joins the antibody preparations previously reported that describe nuclear antigens, or epitopes on antigens, enhanced at mitosis.     

Reversing the effects of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method.

Formalin is a commonly used fixative for tissue preservation in pathology laboratories. A major adverse effect of this fixative is the concealing of tissue antigens by protein cross-linking. To achieve a universal antigen retrieval method for immunohistochemistry under a constant condition, we developed a new method in which the effects of formalin fixation were reversed with citraconic anhydride (a reversible protein cross-linking agent) plus heating. Formalin-fixed, paraffin-embedded tissues from various organs were examined for immunohistochemical localization of a wide variety of antigens. Deparaffinized tissue sections were placed in an electric kitchen pot containing 0.05% citraconic anhydride solution, pH 7.4, and the pot was set at "keep warm" temperature mode of 98C for 45 min. This mode allowed heating the sections at a constant temperature. The sections were then washed in buffer solution and immunostained using a labeled streptavidin-biotin method using an automated stainer. In general, formalin-fixed tissues demonstrated specific immunostainings comparable to that in fresh frozen tissues and significantly more enhanced than after conventional antigen retrieval methods. In particular, even difficult-to-detect antigens such as CD4, cyclin D1, granzyme beta, bcl-6, CD25, and lambda chain revealed distinct immunostainings. Different classes of antigens such as cellular markers and receptors, as well as cytoplasmic and nuclear proteins, consistently produced enhanced reactions. This method provides efficient antigen retrieval for successful immunostaining of a wide variety of antigens under an optimized condition. It also allows standardization of immunohistochemistry for formalin-fixed tissues in pathology laboratories, eliminating inter-laboratory discrepancies in results for accurate clinical and research studies.