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.     

Immunogold-silver staining of mesangial antigen in lowicryl K4M- and LR gold-embedded renal tissue using epipolarization microscopy.

We examined the localization of a glomerular mesangial antigen with a Thy-1.1 monoclonal antibody by epipolarization microscopy (EPI) of silver-enhanced, immunogold-stained renal tissue embedded in LR Gold and Lowicryl K4M, and compared the attributes of these hydrophilic resins. Antigen was well preserved in tissue embedded in both resins. LR Gold-embedded tissue demonstrated excellent immunostaining properties, sectioned more easily, and showed better durability during staining than K4M. Lowicryl K4M-embedded tissue, however, displayed a phenomenon of self-illumination when counterstained with eosin which was not seen with LR Gold. This enabled immunostaining to be precisely related to tissue morphology without the necessity of simultaneous transillumination, which can be problematic when used in combination with EPI because of reflection of incident illumination from sub-stage optical surfaces.     

LR white resin and improved on-grid immunogold detection of vicilin,a pea seed storage protein

LR White resin combined with post-embedding immunogold labelling was used to localize the storage protein vicilin within developing pea seed cotyledons by electron microscopy. Fine structural preservation is comparable to that obtained with Spurr's resin, and antibody labelling is improved. More gold binds to protein bodies, to rough endoplasmic reticulum and to Golgi vesicles, and in addition, vicilin was detected within Golgi cisternae, a site not previously observed.     

Localization of pepsinogen and cellular differentiation in the human gastric mucosa using lowicryl K4M

The localization of pepsinogens (PG A and PG C) was studied intracellularly in human gastric biopsies embedded in Lowicryl K4M, using affinity purified antibodies and protein A-gold. The homogeneous secretory granules of the chief cells contained both PG A and PG C, as was proved in serial sections. Identical reaction was seen in the core of the biphasic mocous neck cell granules, whereas the mantle did not label. Even the rough endoplasmic reticulum (RER) and Golgi complex of the chief- and mucous neck cells contained label. Transitional cells identified by the presence of granules of both chief- and mucous neck cells were seen. This type of mucous neck cell is thought to transform into a chief cell. However an increase of RER that could explain an increase of the pepsinogen production was not observed. A mixture of these granules were also found in morphologically characterized young parietal cells, suggesting a common precursor for these three cell-types. These observations makes the transformation from mucous neck- into chief cells questionable. In conclusion Lowicryl K4M appeared to be a significant improvement compared to the Epon 812. Its shows a better preservation of both cytoplasmic antigens and cellular fine structure. This improvement adds information on the transformation hypothesis. Lowicryl K4M enables us, firstly to distinguish PG A and C synthesizing RER in different types of cell and secondly to recognize immature cells with the characteristics of chief-, mucous neck-, and parietal cells in the fundic gland. Very likely these three cell-types all arise from a common precursor. It is questionable that in normal human gastric mucosa the mucous neck cells transform into chief cells.     

Lowicryl K4M embedding of brain tissue for immunogold electron microscopy

We present methods for embedding brain tissue in Lowicryl K4M embedding medium and localizing antigens using postembedding immunogold techniques. After perfusion fixation with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer, blocks of rat brain were placed in 2% aqueous uranyl acetate for 1 hour, dehydrated in 50%, 70%, and 95% ethanol, infiltrated with Lowicryl/ethanol mixtures (1:2 for 10 min, 1:1 for 15 min) and 100% Lowicryl (20 min and 25 min). Polymerization was carried out under UV light for 24-48 hours at room temperature. Several neural antigens, including three different synaptic vesicle proteins and an enzyme associated with the postsynaptic density, were localized by this technique, indicating that this procedure may have wide applicability.     

Evaluation of LR white resin for histology of the undecalcified rat tibia

Histology of plastic embedded undecalcified bone represents a challenging problem to the histotechnologist. We outline here an exploration of LR White resin as a suitable medium for histologic study of undecalcified rat tibia. A procedure was developed for light microscopy of rat tibia embedded in LR White and sectioned by sawing-grinding technics. The specimens were fixed in 10% neutral buffered formalin or alcohol-acetic acid-formol, dehydrated in ethanol, defatted in chloroform followed by resin infiltration and heat-curing of embedded blocks. The procedure of dehydration, defatting, infiltration, and polymerization can be completed within 10 days. Cold curing with accelerator provided by the manufacturer did not yield superior results compared to blocks cured with heat. Thick sections were obtained using a diamond wire saw, attached to plexiform slides, then ground and polished. Surface staining with Von Kossa silver reagent or toluidine blue revealed satisfactory morphological preservation of the mineralized bone sections. Artifacts like small bubbles appeared occasionally and could not be avoided despite prolonged infiltration or cold curing of blocks. Our method is relatively simple for base-line histologic study of rat tibia. The method offers advantages such as easy adaptability, reliable stainability, contrast, and resolution of bone architecture and marrow cells. Two other embedding media, Micro-Bed resin and Unicryl, were also tested, but produced inferior results.     

Ultrastructural localization of tubulin and actin in polyethylene glycol-embedded rat seminiferous epithelium by immunogold staining

The polyethylene glycol (PEG) method for immunofluorescence localization of cytoskeletal antigens has been extended to the ultrastructural level using glutaraldehyde-fixed tissues and immunogold staining. Semithin sections of fixed tissue embedded in polyethylene glycol are divested of the PEG, exposed to purified antibodies (e.g., antiactin, antitubulin) and anti-IgG-colloidal gold. The sections may be processed by dehydration and critical-point drying, or reembedment in hydrophilic substances. Tubulin is demonstrated in the mitotic spindles of dividing spermatogonia, manchettes, axonemes and centrioles of developing spermatids, and in the Sertoli cell cytoplasm; actin localization is demonstrated in the myoid cells of the tunica propria, and smooth muscle cells of arterioles in the interstitial tissue. The results demonstrate the applicability and versatility of PEG embedding for immunocytochemistry.     

Ultrastructural localization of tubulin and actin in polyethylene glycol-embedded rat seminiferous epithelium by immunogold staining

The polyethylene glycol (PEG) method for immunofluorescence localization of cytoskeletal antigens has been extended to the ultrastructural level using glutaraldehyde-fixed tissues and immunogold staining. Semithin sections of fixed tissue embedded in polyethylene glycol are divested of the PEG, exposed to purified antibodies (e.g., antiactin, antitubulin) and anti-IgG-colloidal gold. The sections may be processed by dehydration and critical-point drying, or reembedment in hydrophilic substances. Tubulin is demonstrated in the mitotic spindles of dividing spermatogonia, manchettes, axonemes and centrioles of developing spermatids, and in the Sertoli cell cytoplasm; actin localization is demonstrated in the myoid cells of the tunica propria, and smooth muscle cells of arterioles in the interstitial tissue. The results demonstrate the applicability and versatility of PEG embedding for immunocytochemistry.     

A method for preserving ultrastructural properties of mitotic cells for subsequent immunogold labeling using low-temperature embedding in LR White resin

The best available approach of biological sample preparation for transmission electron microscopy currently includes cryoimmobilization by high-pressure freezing (HPF) followed by freeze-substitution (FS). This method has been well established for interphase cells; however, a reliable and easy procedure is still missing for mitotic cells especially because of their fragility and sensitivity to treatments. Here, we present a fast and effective method for HPF/automated FS and LR White embedding of mitotic cells which allows for their controlled and reproducible quality processing. It should be useful in various ultrastructural studies on mitotic cells especially in combination with immunocytochemistry.     

Detection of cell proliferation in pig testis and intestine sections using monoclonal anti-bromodeoxyuridine antibody and immunogold silver staining

Summary For the first time a monoclonal antibody against 5-bromodeoxyuridine was used to detect cell proliferation in pig testis and intestine sections. The influence of several parameters such as mode of injection, addition of thymidine biosynthesis inhibitor, tissue fixation, hydrolysis and revelation was examined. The technique of choice consisted in intravenously injecting the animals with 50 mg/kg BUdR added to 10 mg/kg FUdR 2 h before tissue collection and Bouin fixation; hydrolysis of sections was performed by HCl 4N: Ethanol 70° (1:1 v/v); revelation of BUdR was made by a secondary antibody linked to colloidal gold particles, followed by a silver enhancement step. The data were superior when compared to those obtained by direct immunofluorescence and by the PAP technique. The described method is convenient and sensitive, provides an intense nuclear labelling without background and allows simultaneous examination of histology. The advantages over the technique using tritiated thymidine are particularly obvious when fast screening of numerous samples is required or when new experimental protocols are developping.     

LR-White and LR-Gold resins for postembedding immunofluorescence staining of laminin in mouse kidney

Summary This work describes a method for the immunolocalization of laminin on 1m-thick tissue sections using a postembedding immunofluorescence technique. Embedding of unfixed or formaldehyde-fixed mouse renal cortex in either of the acrylic resins LR-White or LR-Gold permitted reliable postembedding immunofluorescence staining for laminin. LR-White was heat-cured at 50°C whereas LR-Gold was polymerized at –25°C. A stronger immunostaining for laminin was obtained from tissue embedded in polymerized LR-Gold compared with the staining from tissue embedded in LR-White. Prerequisites for adequate postembedding immunostaining are the partial dehydration of the tissue (maximum ethanol concentration, 70%) and pepsin treatment of the tissue sections prior to performing the immunostaining reactions.     

Detection of cell proliferation in pig testis and intestine sections using monoclonal anti-bromodeoxyuridine antibody and immunogold silver staining

For the first time a monoclonal antibody against 5-bromodeoxyuridine was used to detect cell proliferation in pig testis and intestine sections. The influence of several parameters such as mode of injection, addition of thymidine biosynthesis inhibitor, tissue fixation, hydrolysis and revelation was examined. The technique of choice consisted in intravenously injecting the animals with 50 mg/kg BUdR added to 10 mg/kg FUdR 2 h before tissue collection and Bouin fixation; hydrolysis of sections was performed by HC1 4N: Ethanol 70 degrees (1:1 v/v); revelation of BUdR was made by a secondary antibody linked to colloidal gold particles, followed by a silver enhancement step. The data were superior when compared to those obtained by direct immunofluorescence and by the PAP technique. The described method is convenient and sensitive, provides an intense nuclear labelling without background and allows simultaneous examination of histology. The advantages over the technique using tritiated thymidine are particularly obvious when fast screening of numerous samples is required or when new experimental protocols are developing.     

Detection of p24 in HIV-1 infected cells embedded in LR White and Lowicryl K4M

Summary In this study we present a postembedding on-grid immunogold labelling procedure for the ultrastructural localization of the HIV-1 core protein p24. HIV-1 infected cells were fixed in 0.1% glutaraldehyde, incompletely dehydrated and embedded in LR White or in Lowicryl K4M. Antigenic sites were detected by incubation of ultrathin sections with primary mouse monoclonal antibody anti-HIV-1 p24, followed by the secondary antibody goat anti-mouse IgG coupled to 10nm gold particles. Antigenicity of p24 was found to withstand the applied fixation and was shown to be preserved in LR White as well as in Lowicryl. The described procedure permits the uncomplicated and easy detection of p24 in HIV-1 infected cells and tissues.     

Cationic gold staining of glomerular anionic sites in archived tissue, reprocessed from paraffin wax into LR gold resin

Summary Glomerular capillary wall anionic sites have been demonstrated by cationic gold staining of archived renal biopsy tissue (up to 10 years old), obtained from six patients, originally embedded in paraffin wax, and subsequently reprocessed into LR gold resin. The staining patterns at pH 2.5 and pH 7.0, demonstrating different glomerular basement membrane (GBM) anionic constituents, were compared in three patients from whom tissue directly processed into LR gold and reprocessed tissue was available. Ultrastructural preservation was poorer and shrinkage artefact greater in paraformaldehyde-lysine periodate (PLP) as opposed to formol saline-fixed reprocessed tissue. However, GBM anionic site expression was well preserved, or even enhanced (lamina rara externa, pH 7.0) in reprocessed tissue, using either fixative. Although it may not be possible to compare subtle changes in anionic site distribution in variously fixed and processed tissues, due to these artefacts, the technique enables retrospective study of charge status in archived material from disease groups in which there are distinct anionic site aberrations.     

Immunoelectron microscopy of acetylcholine receptors and 43 KD protein after rapid freezing, freeze-substitution, and low-temperature embedding in lowicryl K11M

To label intracellular determinants of the acetylcholine receptor and associated cytoplasmic proteins while preserving optimal ultrastructure, we developed a post-embedment labeling technique that uses rapid-frozen specimens and freeze-substitution without chemical fixatives. This procedure has been made possible through the use of a low-temperature resin (Lowicryl K11M) that can be polymerized with UV light at -60 degrees C. Rapid-frozen muscle cells were used to evaluate the preservation of structure, and Torpedo electroplaque cells and purified postsynaptic membranes were used to quantitatively evaluate the labeling specificity, efficiency, and resolution of the technique. The labeling efficiency of seven different monoclonal antibodies (MAb) to the acetylcholine receptor varied from 3-13%; there was a correlation between the degree of efficiency and the number of epitopes with which the antibodies reacted. The resolution of the technique was not sufficient to determine whether the anti-acetylcholine receptor MAb were bound to the cytoplasmic or the extracellular surface, but was sufficient to correctly determine the location of the receptor-associated 43 KD protein on the cytoplasmic surface.     

Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin

Histological techniques are critical for observing tissue and cellular morphology. In this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish embryos embedded in JB-4 plastic resin-a glycol methacrylate-based medium that results in excellent preservation of tissue morphology. In addition, we describe our procedures for staining plastic sections with toluidine blue or hematoxylin and eosin, and show how to couple these stains with whole-mount RNA in situ hybridization. We also describe how to maintain and visualize immunofluorescence and EGFP signals in JB-4 resin. The protocol we outline-from embryo preparation, embedding, sectioning and staining to visualization-can be accomplished in 3 d. Overall, we reinforce that plastic embedding can provide higher resolution of cellular details and is a valuable tool for cellular and morphological studies in zebrafish.     

Demonstration of dopamine in electron-dense synaptic vesicles in the pars intermedia of Xenopus laevis,by freeze substitution and postembedding immunogold electron microscopy

Summary The presence of dopamine in the pituitary of the clawed toad Xenopus laevis was studied by light and electron microscope immunocytochemistry, using pre- and postembedding techniques. Light microscopy showed the presence of an intricate, anti-dopamine-positive fibre network throughout the pars intermedia. In preembedded stained material, dopamine appeared to occur in varicosities which make synaptic contacts with both folliculo-stellate cells and melanotrope cells. Postembedding immunogold staining of freeze-substituted material permitted the localization of anti-dopamine reactivity in electron-dense vesicles in these varicosities. This finding supports the hypothesis that dopamine is involved in the (inhibitory) control of melanotrope cell activity in X. laevis.     

Demonstration of dopamine in electron-dense synaptic vesicles in the pars intermedia of Xenopus laevis, by freeze substitution and postembedding immunogold electron microscopy.

The presence of dopamine in the pituitary of the clawed toad Xenopus laevis was studied by light and electron microscope immunocytochemistry, using pre- and postembedding techniques. Light microscopy showed the presence of an intricate, anti-dopamine-positive fibre network throughout the pars intermedia. In preembedded stained material, dopamine appeared to occur in varicosities which make synaptic contacts with both folliculo-stellate cells and melanotrope cells. Post-embedding immunogold staining of freeze-substituted material permitted the localization of anti-dopamine reactivity in electron-dense vesicles in these varicosities. This finding supports the hypothesis that dopamine is involved in the (inhibitory) control of melanotrope cell activity in X. laevis.     

The immunogold staining technique for the measurement of lymphocyte subpopulations in bronchoalveolar lavage fluid

The immunogold staining technique was evaluated for use in the identification of lymphocyte subsets in bronchoalveolar lavage fluid. The results obtained compared favorably to the identification of lymphocytes by the E-rosette method, the acid alpha-naphthyl-acetate esterase (ANAE) stain and immunofluorescence microscopy. The main advantages of the immunogold staining method include improved cellular detail and the attainment of permanent preparations, allowing for reassessment, intralaboratory comparison and the performance of further histochemical techniques if desired.