Streptococcal protein G-gold complex: comparison with staphylococcal protein A-gold complex for spot blotting and immunolabeling

Protein G, a cell wall protein isolated from human group G streptococci strain G148, binds in a similar manner as protein A from Staphylococcus aureus to the Fc portion of IgG molecules. Indeed, protein G has been proposed as a superior Fc binding protein due to its broader species reactivity. Thus, we have prepared a complex of protein G with particles of colloidal gold and determined its applicability for spot-blot analysis and postembedding immunolabeling by comparing it with protein A-gold complex. By spot-blot analysis no difference in binding of protein G-gold or protein A-gold to IgG molecules from a whole spectrum of animal species was observed. Moreover, using rabbit, sheep, or goat anti-rat albumin antibodies to detect nitrocellulose-immobilized rat albumin or antigenic sites in paraffin and Lowicryl K4M thin sections from rat liver, no difference was found with protein G-gold or protein A-gold. Similarly, no difference in binding to protein G-gold or protein A-gold was observed with a battery of monoclonal antibodies. However, in contrast to expectations, protein A-gold reacted well with both sheep and goat IgG molecules; indeed, for the light and electron microscopic localization of albumin with sheep or goat antibodies it was as efficient as protein G-gold. These results demonstrate, therefore, that both protein G-gold and protein A-gold are useful second step reagents for immunolabeling and that protein G-gold was not a superior probe in the systems tested.     

Detection of Lignin Peroxidase and Xylanase by Immunocytochemical Labeling in Wood Decayed by Basidiomycetes

The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra-and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay. Lignin peroxidase was localized at sites within the cell wall where electron-dense areas of the lignified cell wall layers remained. In wood decayed by Trametes versicolor, lignin peroxidase was located primarily along the surface of eroded cell walls. No lignin peroxidase was evident in brown-rotted wood, but slight labeling occurred within hyphal cells. Use of polyclonal antisera to xylanase followed by immunogold labeling showed intense labeling on fungal hyphae and surrounding slime layers and within the woody cell wall, where evidence of degradation was apparent. Colloidal-gold-labeled xylanase was prevalent in wood decayed by all fungi used in this study. Areas of the wood with early stages of cell wall decay had the greatest concentration of gold particles, while little labeling occurred in cells in advanced stages of decay by brown or white rot fungi.     

Uukuniemi virus maturation: immunofluorescence microscopy with monoclonal glycoprotein-specific antibodies.

Monoclonal antibodies directed against Uukuniemi virus glycoproteins G1 and G2 in combination with polyclonal antibodies against the nucleoprotein (N) were used to study the maturation of the virus in Golgi complexes of infected chicken embryo fibroblasts and BHK cells. Of 25 monoclonal antibodies obtained, 10 were shown to be G1 specific and 15 were shown to be G2 specific by immunoblotting and immunoprecipitation. In double-staining experiments, some of the monoclonal antibodies gave similar distributions of fluorescence as compared with the staining obtained from polyclonal rabbit anti-G1-G2 antibodies. Others, however, preferentially stained either the glycoproteins in the Golgi complex or those at the cell surface. This may indicate that the glycoproteins underwent conformational changes during their transport. Uukuniemi virus infection resulted in the vacuolization of the membranes of Golgi complexes where the maturation of the virus was taking place. Double-staining experiments with monoclonal antibodies which preferentially stained the Golgi-associated viral glycoproteins and with anti-N polyclonal rabbit antiserum showed a correlation between the progressive vacuolization of the Golgi complex and the accumulation of viral nucleoprotein in the Golgi region, suggesting that a morphological alteration of the Golgi complex may be a prerequisite for intracellular maturation of the virus. Treatment of Uukuniemi virus-infected cells with tunicamycin, a drug which inhibits N-linked glycosylation, resulted in the accumulation of both glycoproteins at an intracellular location, apparently representing the endoplasmic reticulum. Double-staining experiments showed a parallel accumulation of nucleoprotein at these sites, indicating that local accumulation of glycoproteins is required for nucleoprotein binding to intracellular membranes.     

Subcellular localization of tissue polypeptide antigen and cytokeratins in epithelial cells (salivary ad mammary glands). Combined use of the cryoultramicrotomy and the protein A-gold technique

Epithelial cells from various sites and at various stages of differentiation reveal distinct cytokeratin polypeptide patterns. WE have localized these heterogeneous elements at the subcellular level in human salivary glands and in a solid tumor of the breast using a monoclonal and a polyclonal antibody against cytokeratin, and an antibody against tissue polypeptide antigen (TPA) which seems to be related to some cytokeratins. Labeling by the cytokeratin antibodies was more intense in squamous and duct cells than in acinar cells. The TPA:B1 antibody reacted predominantly with duct cells and to a lesser extent with acinar and squamous cells. A precise evaluation of the labeling pattern and a well-preserved cell structure appeared to be important factors in obtaining more detailed information about intermediate filament proteins. The cryoultramicrotomy and the protein A-gold technique are suitable for these studies.     

The detection of substructures within proteoglycan molecules. Electron-microscopic immuno-localization with the use of Protein A-gold.

Proteoglycan monomers from pig laryngeal cartilage were examined by electron microscopy with benzyldimethylalkylammonium chloride as the spreading agent. The proteoglycans appeared as extended molecules with a beaded structure, representing the chondroitin sulphate chains collapsed around the protein core. Often a fine filamentous tail was present at one end. Substructures within proteoglycan molecules were localized by incubation with specific antibodies followed by Protein A-gold (diameter 4 nm). After the use of an anti-(binding region) serum the Protein A-gold (typically one to three particles) bound at the extreme end of the filamentous region. A small proportion of the labelled molecules (10-15%) showed the presence of gold particles at both ends. A monoclonal antibody specific for a keratan sulphate epitope (MZ15) localized a keratan sulphate-rich region at one end of the proteoglycan, but gold particles were not observed along the extended part of the protein core. This distribution was not changed by prior chondroitin AC lyase digestion of the proteoglycan. Localization with a different monoclonal antibody to keratan sulphate (5-D-4) caused a change in the spreading behaviour of a proportion (approx. 20%) of the proteoglycan monomers that lost their beaded structure and appeared with the chondroitin sulphate chains projecting from the protein core. In these molecules the Protein A-gold localized antibody (5-D-4) along the length of the protein core whereas in those molecules with a beaded appearance it labelled only at one end. Labelling with either of the monoclonal antibodies was specific, as it was inhibited by exogenously added keratan sulphate. The differential localization achieved may reflect structural differences within the proteoglycan population involving keratan sulphate and the protein core to which it is attached. The results showed that by this technique substructures within proteoglycan molecules can be identified by Protein A-gold labelling after the use of specific monoclonal or polyclonal antibodies.     

Immunoglobulin G specifically binding plant N-glycans with high affinity could be generated in rabbits but not in mice

Xylosylated and core alpha1,3-fucosylated N-glycans from plants are immunogenic, and they play a still obscure role in allergy and in the field of plant-made protein pharmaceuticals. We immunized mice to generate monoclonal antibodies (mAbs) binding plant N-glycans specifically via the epitope containing either the xylose or the core alpha1,3-fucose residue. Splenocytes expressing N-glycan-specific antibodies derived from C57BL/6 mice previously immunized with plant glycoproteins were preselected by cell sorting to generate hybridoma lines producing specific antibodies. However, we obtained only mAbs unable to distinguish fucosylated from xylosylated N-glycans and reactive even with the pentasaccharide core Man3GlcNAc2. In contrast, immunization of rabbits yielded polyclonal sera selectively reactive with either fucosylated or xylosylated N-glycans. Purification of these sera using glyco-modified neoglycoproteins coupled to a chromatography matrix provided polyclonal sera suitable for affinity determination. Surface plasmon resonance measurements using sensor chips with immobilized glyco-modified transferrins revealed dissociation constants of around 10(-9) M. This unexpectedly high affinity of IgG antibodies toward carbohydrate epitopes has repercussions on our conception of the binding strength and significance of antiglycan IgE antibodies in allergy.     

Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.     

Electron microscopic localization of glutamate dehydrogenase in rat liver mitochondria by an immunogold procedure and monoclonal and polyclonal antibodies

Glutamate dehydrogenase (GDH) was localized in rat liver by indirect electron microscopic immunogold, using different sizes of gold particles and monoclonal and polyclonal antibodies. Using the protein A-gold technique in double immunocytochemical experiments, both antibodies, at their optimal dilutions, gave similar results. A novel assessment of the distribution of GDH was made by measurements of the number of gold particles per square micrometer of cross-sectional images of individual mitochondria. The data indicate intracellular homogeneity among mitochondria in individual parenchymal cells. The enzyme is almost absent in non-parenchymal cells. Finally, GDH was found mainly in association with the mitochondrial inner membrane.     

Wall ingrowths in epidermal transfer cells of Vicia faba cotyledons are modified primary walls marked by localized accumulations of arabinogalactan proteins

Despite the importance of transfer cells in enhancing nutrient transport in plants, little is known about how deposition of the complex morphology of their wall ingrowths is regulated. We probed thin sections of mature cotyledon epidermal transfer cells of Vicia faba with affinity probes and antibodies specific to polysaccharides and glycoproteins, to determine the distribution of these components in their walls. Walls of these transfer cells consist of the pre-existing primary wall, a uniformly deposited wall layer and wall ingrowths which are comprised of two regions; an electron-opaque inner region and an electron-translucent outer region. The primary wall reacted strongly with antibodies against esterified pectin, xyloglucan, the side chains of rhamnogalaturonan-1 and a cellulase-gold affinity probe. The electron-opaque inner region of wall ingrowths displayed a similar labeling pattern to that of the primary wall, showing strong cross-reactivity with all antibodies tested, except those reacting against highly de-esterified pectins. The electron-opaque outer layer of developmentally more mature wall ingrowths reacted strongly with anti-callose monoclonal and polyclonal antibodies, but showed no reaction for pectin or xyloglucan antibodies or the cellulase-gold affinity probe. The plasma membrane-wall interface was labeled strongly with anti-arabinogalactan protein (AGP) antibodies, with some AGP-reactive antibodies also labeling the electron-translucent zone. Nascent wall ingrowths were labeled specifically with AGPs but not anti-callose. A reduction in wall ingrowth density was observed when developing transfer cells were exposed to beta-d-glucosyl Yariv reagent compared with controls. Our results indicate that wall ingrowths of transfer cells are primary wall-like in composition and probably require AGPs for localized deposition.     

Identification and partial characterization of human platelet C1q binding sites

We recently showed that human blood platelets bind the complement component, C1q, and mAb directed against lymphoblastoid C1q receptors in a specific and saturable manner. To identify and further characterize platelet C1q binding sites, human platelets were washed, solubilized in Triton X-100 and either subjected to SDS-PAGE and Western blotting by using monoclonal (II1/D1) and polyclonal antibodies recognizing C1qR on lymphoblastoid cells, or applied to a C1q-Sepharose affinity column under low ionic strength conditions (20 mM NaCl). Adherent proteins were eluted with buffer containing 300 mM NaCl. Western blotting with monoclonal and polyclonal antibodies directed against C1qR showed exclusive reactivity with a 67,000 m.w. protein possessing intrachain disulfide bonds. SDS-PAGE of C1q-Sepharose eluates also revealed the presence of a 67,000 protein which was accompanied to varying degrees by a 94,000 constituent. Because similar m.w., 125I-labeled proteins were recovered from C1q-Sepharose columns to which lysed, surface-labeled platelets had been applied, both 94,000 and 67,000 components appear to be platelet membrane constituents. The 94,000 and 67,000 species, however, appear to be antigenically distinct. The 94,000 protein was immunoprecipitated by polyclonal antibodies against platelet membrane glycoprotein IIIa but not polyclonal antibodies against C1qR, whereas the 67,000 protein was immunoprecipitated exclusively by the polyclonal anti-C1qR antibody. The 67,000 protein thus appears to represent platelet C1q binding sites resembling C1qR on lymphoblastoid cells.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.     

Subunit composition of the molybdate-stabilized "8-9 S" nontransformed estradiol receptor purified from calf uterus

The structure of the calf uterus nontransformed molybdate-stabilized estradiol receptor (ER) has been investigated using affinity labeling with tamoxifen aziridine and several monoclonal antibodies directed either against the steroid binding protein (Mr approximately 65,000) or against the heat shock protein of Mr approximately 90,000 (hsp 90). The purification was performed using affinity chromatography and a DEAE-Sephacel column. The [3H] estradiol-ER complex was obtained as a well-defined radioactive peak, the specific activity varying between 1,600 and 3,400 pmol/mg of protein. The purified ER sediments in glycerol gradients at 9.4 S +/- 0.2 (n = 5) and at 8.1 S +/- 0.2 (n = 15) in a 0.15 M KCl containing gradient ("8-9 S" ER). From a measured Stokes radius of 7.4 +/- 0.2 nm (n = 12), an Mr of approximately 300,000 has been calculated. Studies of the purified 8-9 S ER by glycerol gradient centrifugation and by "twin antibody" assay with the JS34/32 anti-ER monoclonal antibody suggest the presence of two binding subunits in the nontransformed molecular complex. Results of immunological analysis with polyclonal and several monoclonal antibodies against hsp 90 suggest the association of two molecules of this protein to the two steroid binding subunits. In high salt medium (0.4 M KCl), the purified ER sediments at 5.2 +/- 0.3 (n = 8), has a Stokes radius of 5.7 nm +/- 0.1 (n = 2) and the Mr is approximately 129,000, values expected for a homodimer consisting of two hormone-binding subunits (Mr approximately 65,000), a result confirmed by glycerol gradient centrifugation experiments, using the monoclonal antibody JS34/32. The relationship between the nontransformed 8-9 S ER and the transformed 5 S-ER forms are discussed, the simplest possibility being the release of the already formed homodimeric ER from 8-9 S ER during transformation.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

Abstract. A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.     

Specificity of the localization of transforming growth factor-alpha immunoreactivity in colon mucosa.

Transforming growth factor-alpha (TGF-alpha) plays an important role in gastrointestinal pathophysiology. However, the exact location of its expression in the intestine is still controversial. This study systematically compared the localization of TGF-alpha immunoreactivity in frozen or fixed human colon using three different antibodies and examined specificity of antibodies by using tissues from TGF-alpha knockout mice and by Western blotting. Consistent with the mRNA distribution revealed by in situ hybridization, a similar staining pattern was obtained in frozen sections by all three antibodies, localizing on the surface and along the crypt epithelium. In paraffin sections, although the polyclonal antibodies (raised against recombinant human or rat TGF-alpha) gave minimal staining, the monoclonal antibody (against C-terminal peptide of human TGF-alpha) still gave intense staining on the surface and upper crypt epithelium. By using specimens from TGF-alpha knockout mice in immunostaining and Western blotting, the polyclonal antibodies were shown to be specific. In contrast, specificity of the monoclonal antibody was in doubt in rodent tissues because it gave similar detection between wild-type and knockout mice in both analyses, indicating its crossreaction to non-TGF-alpha molecules. In conclusion, frozen sections and antibodies raised from recombinant TGF-alpha should be used for TGF-alpha immunohistochemistry in the colon.     

High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.     

Modification of the protein A-gold immunocytochemical technique for the enhancement of its efficiency

A modification in the protein A-gold immunocytochemical technique has been introduced for amplification of the labeling. This modification consists of performing additional incubation steps with an anti-protein A antibody and the protein A-gold complex. The original antigen-antibody-protein A-gold complex was further incubated with an antibody directed against protein A and then, in a fourth step, again with protein A-gold. This multiple-step protocol results in significant enhancement of the original signal. The modified technique can be applied to either light or electron microscopy protein A-gold immunocytochemistry. The advantage of such an approach is double: it allows for either amplification of the labeling when the original signal is of low intensity or use of highly diluted antibody solutions. The modification introduced was thus found to significantly enhance the efficiency of the technique.     

Comparison of LR white resin, Lowicryl K4M and Epon postembedding procedures for immunogold staining of actin in the testis

The efficiency of various postembedding procedures for actin immunogold detection was compared using testicular tissue as a model. Whatever the fixative, testes embedded in LR White resin or in Lowicryl K4M showed few differences as regard ultrastructural preservation and gave similar actin antigenicity preservation. A purified polyclonal antibody (IgG) and a monoclonal antibody (IgM) visualized with gold secondary antibody yielded high labeling intensity whereas the IgG-protein-A gold association was less efficient. Crude antisera gave a low specific staining/background ratio. Samples of testes, fixed in different conditions, were also embedded in Epon, omitting propylene oxide and lowering polymerization temperature to 40 degrees-50 degrees C. This slight modification improved ultrastructural preservation which was better than with hydrophilic resins, as well as made possible immunogold detection of actin though antigenicity preservation was lesser than with these resins. Thus, in Epon embedded samples actin labeling, using IgG antiactin-gold secondary antibody, was similar to that observed after hydrophilic resin-protein-A gold procedures. In addition to actin labeling of various somatic cells it was confirmed that actin is a consistent component of the subacrosomal space of spermatids during the greater part of spermiogenesis in rat.     

单克隆抗体与多克隆抗体配对ELISA方法比较

以人绒毛膜促性腺激素(HCG)为抗原,制备出对HCG的多克隆抗体和特异性单克隆抗体,并进行抗体纯化和特性分析,利用辣根过氧化物酶(HRP)分别对其进行了标记.采用双抗夹心ELISA试验,探讨了多克隆抗体与单克隆抗体配对的若干事项.结果表明,利用单克隆抗体和酶标多克隆抗体配对,并用含动物血清的稀释液稀释酶标抗体,可实现对检测原的高特异性和高灵敏度检测.     

Commercial polyclonal and monoclonal histostaining PAP kits

Summary Universal, polyclonal and monoclonal immunoperoxidase staining kits from BioGenex, Dako and Ortho were employed for the localization of antigens such as gastrin, prostate specific antigen, IgA, IgG, AFP and CEA in histological sections from formaldehyde fixed and paraffin embedded human specimens. The kit components were controlled by immunohistological and serological assays and were also compared with self-prepared reagents. In connection with specific primary antibodies, universal/basic kits gave reliable localization of defined antigens. The optimal concentration of the primary antibodies had to be established by dilution experiments. In the case of polyclonal kits, typical antigen localization was obtained in selected tissue sections with all the respective kits. CEA kits also stained strongly NCA molecules present in organs such as colon, stomach and liver. BioGenex polyclonal kits gave almost stronger stainings than kits from Dako and Ortho. Irrespective of which kit from different commercial sources is used, development of peroxidase activity with AEC/H₂O₂ often had to be stopped far below the recommended incubation time of 40 min or overstaining with color change from reddish to muddy green occurred. The latter was attributed to insufficiently balanced kit reagents, an interpretation wich was supported by quantitative serological studies. Sensitivity of immunohistological reactivity was much enhanced by pretreatment of tissue sections with Pronase. Thus, stronger immunostainings and larger numbers of positive cells were detected than in conventionally rehydrated sections. Incubation of sections with self-prepared primary antibodies, linking antibodies and PAP complexes gave essentially the same antigen localization as with commercial kits, but antibodies isolated by our affinity chromatography led to a better staining contrast with absence of nonspecific background. The advantage of monoclonal over polyclonal kits was the background-free staining of sections. Other-wise, antigens were localized in the same cell types, although cellular reactivity was usually less intense than with polyclonal antibodies. This, however, could be overcome by Pronase treatment of the sections prior to incubation.     

Comparison of LR white resin,lowicryl K4M and Epon postembedding procedures for immunogold staining of actin in the testis

Summary The efficiency of various postembedding procedures for actin immunogold detection was compared using testicular tissue as a model. Whatever the fixative, testes embedded in LR White resin or in Lowicryl K4M showed few differences as regard ultrastructural preservation and gave similar actin antigenicity preservation. A purified polyclonal antibody (IgG) and a monoclonal antibody (IgM) visualized with gold secondary antibody yielded high labeling intensity whereas the IgG-protein-A gold association was less efficient. Crude antisera gave a low specific staining/background ratio. Samples of testes, fixed in different conditions, were also embedded in Epon, omitting propylene oxide and lowering polymerization temperature to 40°–50° C. This slight modification improved ultrastructural preservation which was better than with hydrophilic resins, as well as made possible immunogold detection of actin though antigenicity preservation was lesser than with these resins. Thus, in Epon embedded samples actin labeling, using IgG antiactin-gold secondary antibody, was similar to that observed after hydrophilic resin-protein-A gold procedures. In addition to actin labeling of various somatic cells it was confirmed that actin is a consistent component of the subacrosomal space of spermatids during the greater part of spermiogenesis in rat.