Pre-embedding Staining Research Articles

Hematopoietic bone marrow cells participate in endothelial, but not epithelial or mesenchymal cell renewal in adult rats

The extent to which bone marrow (BM) contributes to physiological cell renewal is still controversial. Using the marker human placental alkaline phosphatase (ALPP) which can readily be detected in paraffin and plastic sections by histochemistry or immunohistochemistry, and in ultrathin sections by electron microscopy after pre-embedding staining, we examined the role of endogenous BM in physiological cell renewal by analysing tissues from lethally irradiated wild-type inbred Fischer 344 (F344) rats transplanted (BMT) with unfractionated BM from ALPP-transgenic F344 rats ubiquitously expressing the marker. Histochemical, immunohistochemical and immunoelectron microscopic analysis showed that the proportion of ALPP+ capillary endothelial cells (EC) profoundly increased from 1 until 6 months after BMT in all organs except brain and adrenal medulla. In contrast, pericytes and EC in large blood vessels were ALPP–. Epithelial cells in kidney, liver, pancreas, intestine and brain were recipient-derived at all time-points. Similarly, osteoblasts, chondrocytes, striated muscle and smooth muscle cells were exclusively of recipient origin. The lack of mesenchymal BM-derived cells in peripheral tissues prompted us to examine whether BMT resulted in engraftment of mesenchymal precursors. Four weeks after BMT, all haematopoietic BM cells were of donor origin by flow cytometric analysis, whereas isolation of BM mesenchymal stem cells (MSC) failed to show engraftment of donor MSC. In conclusion, our data show that BM is an important source of physiological renewal of EC in adult rats, but raise doubt whether reconstituted irradiated rats are an apt model for BM-derived regeneration of mesenchymal cells in peripheral tissues.

NO-sensitive guanylyl cyclase is expressed in pericytes but absent from endothelial cells in the murine lung

Background In the lung, nitric oxide (NO) regulates vascular tone and permeability via its receptor NO-sensitive guanylyl cyclase (NO-GC). Although data from vascular beds of other species imply that NO-GC is expressed in microvascular endothelial cells, definitive information of its cellular expression in the alveolar region is lacking. This study aimed to clarify the expression of NO-GC in the lung by using an antibody specific for the b1 subunit of NO-GC as judged by absence of staining in NO-GCdeficient mice [1,2]. Immunohistochemistry was performed in murine lungs using 200 µm thick precision cut lung slices (PCLS) and frozen sections for preembedding staining on the EM level. In addition to NO-GC, the expression of phosphodiesterase 5 (PDE5) was determined by immunohistochemistry. Furthermore, cGMP production was visualized using a cGMP-specific antibody after NO stimulation of PCLS in the absence and presence of the PDE5 inhibitor sildenafil. Activity and expression of NO-GC in cultured microvascular murine lung endothelial cells were determined by radioimmunoassay of NO-induced cGMP synthesis and Western blotting, respectively. Results NO-GC immunoreactivity was detected in vascular smooth muscle cells as anticipated from functional studies. In the alveolar region, however, NO-GC staining was restricted to cells in the alveolar wall; these cells were distinct from endothelial cells and exhibited highly branched processes. These processes were in close contact with endothelial cells. Close to the pleural surface where alveolar capillaries tend to be more elongated, NO-GC-immunoreactive cells had the classical morphology of pericytes. Electron microscopy revealed that NOGC-immunoreactive processes of highly branched cells shared the basement membrane with endothelial cells which is characteristic for pericytes. Cells with the same morphology as NO-GC-immunoreactive cells exhibited PDE5 immunoreactivity. Indeed, in the presence of sildenafil, NO stimulation of PCLS resulted in cGMP-specific immunostaining in the highly branched NO-GC-immunoreactive cells. Stimulation of PCLS in the absence of PDE inhibitor did not result in any detectable cGMP. Endothelial cells did not reveal any immunoreactivity for NO-GC, cGMP or PDE5. In addition, NO-GC was not detected in cultured murine lung microvascular endothelial cells by Western blotting and, in line with this, NOinduced cGMP synthesis could not be measured in these cells. Conclusion Our results indicate that NO-GC is expressed in pericytes but not in endothelial cells in the mouse lung. It is therefore conceivable that NO-driven changes in lung microvascular permeability are mediated by pericytes.

Immunocytochemistry of the amphibian embryo--from overview to ultrastructure.

Amphibian embryos are standard research objects to study pattern formation and morphogenesis. Due to their external development and robust nature, experimental manipulations such as microinjections or transplantations can be easily performed. However, most immunocytochemical approaches addressing the specific localization of proteins are hampered by the fragility of the large and yolky embryonic cells which render high resolution staining difficult. Immunocytochemical data are therefore often restricted to either overall patterns in whole embryo preparations or to immunofluorescent localization with limited resolution on sections. High resolution or ultrastructural protein localization data are rare and can be achieved only with time consuming procedures. Here, a comparative study of immunocytochemical methods suitable for light and electron microscopy using different kinds of plastic resins is presented. Three main approaches are described: preembedding staining of whole embryos, postembedding staining of ultrathin sections and preembedding staining of vibratome sections. All the procedures are designed to study protein expression in early amphibian embryos en gros as well as en detail and the described techniques are suitable to combine two or three levels of resolution on the very same biological specimen. Examples are presented and advantages and disadvantages of the different protocols are discussed.

Sulfatide storage in visceral organs of arylsulfatase A-deficient mice.

The inherited deficiency of arylsulfatase A (ASA) in humans causes lysosomal accumulation of sulfatides in visceral organs and in the nervous system and leads to wide-spread demyelination (metachromatic leukodystrophy, MLD). ASA-deficient mice have previously been generated by means of targeted gene disruption. In the present study, visceral organs of ASA-deficient mice were investigated. A simple technique for the histochemical detection of accumulated sulfatides was elaborated using pre-embedding staining with alcian blue. The gall bladder, intrahepatic bile ducts, exocrine pancreatic ducts, respiratory epithelium and, with low degree, testicular Sertoli cells, showed sulfolipid storage. The storage pattern in the kidney will be described in a separate publication. Hepatocytes, pancreatic islets, adrenal glands, and gastric epithelium were unaffected. Ultrastructurally, the intralysosomal storage material displayed parallel and concentric lamellar patterns. Apart from some differences, the topographic distribution of the sulfatide storage resembled that in human MLD. In addition to being an animal model of the human disease, the ASA-deficient mouse may be useful for investigating the cell biology of sulfolipids in visceral organs.

Cytochemical and immunocytochemical methods for electron microscopic detection of glucose-6-phosphate dehydrogenase in brain areas

This paper reports on protocols for the cytochemical and immunocytochemical determination of the glucose-6-phosphate dehydrogenase (G6PD) in brain areas by electron microscopy (EM). The cytochemical assay consists of a pre-embedding staining of small and flat tissue blocks, which were first mildly fixed and then floated in a staining mixture based on the reduction of tetrazolium salts by NADPH. Tissue blocks were then washed, post-fixed in OsO 4, dehydrated through graded ethanol concentrations and embedded in resin. Ultrathin sections were then obtained and observed at the EM. The immunocytochemical technique was performed on completely fixed tissues of perfused animals. After the tissue embedding in resin, ultrathin sections were obtained and treated with a primary anti-erythrocyte G6PD antibody, produced and purified in our laboratory. The immunostaining was performed with secondary gold-conjugated antibody. Gold grains were well evident by EM analysis thus revealing the G6PD protein in the subcellular compartments. These protocols are useful to detect peculiar populations of neurons which express high levels of G6PD to sustain processes of neural plasticity in some brain areas. Themes: Development and regeneration Topics: Cell division and differentiation

Platelet activating factor receptor (PAF-R) is found in a large endosomal compartment in human umbilical vein endothelial cells.

In previous studies, we have localized the platelet activating factor receptor (PAF-R) in situ on the surface of the endothelium in a number of microvascular beds without providing information on its intracellular location. In the present study, we used human umbilical vein cells (HUVECs) as a model to immunolocalize PAF-R by light and electron microscopic procedures. We raised two different polyclonal antibodies against synthetic peptides of the C- and N-terminal of PAF-R and used them for immunolocalization studies. By immunofluorescence, we found that the anti-C-terminal antibody (CPAF-R) stains an extensive intracellular tubular network. By electron microscopy, using a preembedding staining procedure, we detected PAF-R on the surface of the plasmalemma in a staining pattern similar to that described on microvascular endothelia in situ, but at a considerably lower density. Immunogold labeling of thin frozen sections revealed the presence of PAF-R on the plasmalemma, and especially in an extensive network of tubular-vesicular elements and vesicles associated with it. No detectable amounts of PAF-R were found in the endoplasmic reticulum (ER) or in Golgi cisternae. Double immunofluorescence labeling with antibodies for compartment marker proteins and PAF-R revealed that PAF-R localizes in an endosomal compartment. Confocal microscopy showed that PAF-R colocalizes in this compartment together with the transferrin receptor (Tf-R) and the thrombin receptor (TH-R), but it also showed that the colocalization was partial rather than complete. These findings suggest that the endosomal network is either discontinuous or, conversely, that the proteins in its membrane do not have a fully randomized distribution.

Pre-embedding staining for GAD67 versus postembedding staining for GABA as markers for central GABAergic terminals.

Pre-embedding immunocytochemistry for the active form of glutamate decarboxylase (GAD67) and postembedding staining for gamma-aminobutyric acid (GABA) were compared as markers for central GABAergic terminals in the phrenic motor nucleus, in which phrenic motor neurons had been retrogradely labeled with cholera toxin B-horseradish peroxidase. Nerve terminals with or without GAD67 immunoreactivity were identified in one ultrathin section. GABA was localized with immunogold in an adjacent section after etching and bleaching. GABA labeling density was assessed over 519 GAD67-positive and GAD67-negative nerve terminals in the phrenic motor nucleus. Frequency histograms showed that statistically higher densities of gold particles occurred over most GAD67-positive terminals. However, some GAD67-negative terminals also showed high densities of gold particles, and some GAD67-positive terminals showed low densities. Preabsorption of the anti-GABA antibody with a GABA-protein conjugate, but not with other amino acid-protein conjugates, significantly reduced gold labeling over both GAD67-positive and GAD67-negative terminals. These results show that the presence of GAD67 immunoreactivity correlates strongly with high densities of immunogold labeling for GABA in nerve terminals in the phrenic motor nucleus. Preabsorption controls indicate that authentic GABA was localized in the postembedding labeling procedure. Only a small proportion of intensely GABA-immunoreactive terminals lack GAD67, suggesting that both GAD67 and GABA are reliable markers of GABAergic nerve terminals.

The ultrastructure of anionic sites in rat articular cartilage as revealed by different preparation methods and polyethyleneimine staining.

The ultrastructure of anionic sites in the middle layer of rat articular cartilages was studied by two methods, the quick-freezing and deep-etching method, and the quick-freezing and freeze-substitution method. The anionic sites were visualized with a cationic tracer, polyethyleneimine. They were also compared with those revealed in tissues subjected to conventional fixation, such as pre-embedding or post-embedding. With the deep-etching method, three-dimensional meshwork structures were observed more clearly in the extracellular matrix compared with those seen in conventional ultrathin sections. In combination with polyethyleneimine staining, in which no chemical contrast was needed for visualization of anionic sites, numerous stained particles were detected around filaments in the extracellular matrix, indicating that they were anionic sites consisting mainly of proteoglycans. With the pre-embedding method and polyethyleneimine staining, the shapes of aggregated stained particles varied with different preparation procedures, including chemical fixation and contrasting. The fine meshworks were also observed with the post-embedding method and polyethyleneimine staining. It is suggested that such images of anionic sites, as revealed by the deep-etching method and the post-embedding polyethyleneimine-staining method with low-temperature dehydration, are probably closer to native states than those revealed by the conventional pre-embedding polyethyleneimine-staining method.

Immunogold-Silver Staining (IGSS) of Agarose Embedded (NCP) Bvdv-Infected Cell Suspensions

The use of pre-embedding immunogold-silver staining (IGSS) in electron microscopy of cell culture monolayers has been reported by various authors and its usefulness to localize antigens has been documented. Embedding cell suspensions or cell fractions for IGSS can require time-consuming centrifugation steps. Embedding cell suspensions in agarose simplifies IGSS processing and embedding procedures.Bovine turbinate cells (BTs) were grown to confluence in 25cm3culture flasks. BTs were infected with a non-cytopathic strain (ncp) of bovine viral diarrhea virus (BVDV) while control cells were sham inoculated. At 18 h pi, cells were detached with trypsin. All following procedures were carried out at RT. The cells were fixed for 30 min in 2.0% paraformaldehyde + 0.2% glutaraldehyde in 0.1 M. phosphate buffer, pH 7.4, and rinsed 3 times in 0.1 M phosphate buffer, pH 7.4 (each centrifuged for 10 min at 300g). The cells were resuspended in 1 ml of buffer, transferred to an eppendorf tube and centrifuged for 10 min at 300g. The excess buffer was removed, 1-2 drops of warm, molten 1.0% agarose added, and the mixture centrifuged for 1 min at 300g to pellet the cells to the tip of the tube. The solidified cell-agarose mixture was removed, cut into <0.5mm3pieces, and placed in phosphate buffer.

Embryonic form of N-CAM and development of the rat corticospinal tract; immuno-electron microscopical localization during spinal white matter ingrowth

The neural cell adhesion molecule N-CAM has been proposed to function in the guidance of outgrowing axons in the peripheral and central nervous system. Light microscopic observations have shown that the embryonic form of N-CAM (200–230 kDa) is present in the ventralmost part of the dorsal funiculus during corticospinal tract (CST) ingrowth in the first postnatal week (Joosten, Dev. Brain Res., 78 (1994) 226–236). Here, the subcellular localization of the embryonic form of N-CAM (E-NCAM) is determined by pre-embedding staining on vibratone sections and by postembedding immunogold-labelling on Epon embedded spinal cord sections. The electron microscopical observations indicate that E-NCAM is present on the outer membrane of CST growth cones as well as other unmyelinated axons which are present in the ventralmost part of the dorsal funiculus. Furthermore, E-NCAM is localized in an irregular patchy way on the outer side of the axonal membrane of small unmyelinated, later arriving CST axons. From these results it may be deduced that E-NCAM is involved in CST tract formation through guidance of outgrowing pioneer CST growth cones along other unmyelinated axons and through mediation of axon fasciculation of later arriving CST axons.

Cell-surface glycoconjugate layer in the tubotympanic mucosa of the guinea pig as revealed by wheat germ agglutinin/gold labelling.

To evaluate the protective function of the mucous blanket (MB) against lectin substances, we examined at the ultrastructural level whether intraluminal colloidal gold-labelled wheat germ agglutinin (WGA) could enter the MB-covered epithelial cell surface of the guinea pig tubotympanic mucosa. Post-embedding staining with WGA/gold on thin tissue sections was done in parallel for comparison. The cell surface glycoconjugate of the eustachian tubal and transitional epithelium had a typical bilayered structure: the outer MB and the microvilli-associated glycocalyx (MAG), which were interposed by the interciliary fluid zone. In squamous epithelium of the distal middle ear, the MB adhered to the MAG, thereby forming a monolayered coat of glycoconjugates at the cell surface. In the pre-embedding staining, WGA/gold did not bind with the MB and MAG in the eustachian tube, and exclusively bound with MB in the transitional area. Direct binding was also found with MAG and the apical plasmic membrane in the squamous epithelium. These findings indicate that MAG is occluded by MB lined with the interciliary fluid zone for luminal access of lectin at the proximal lumina of the tubotympanic epithelium. It is also suggested that MB existing at two sites possesses a different WGA-binding capacity: shielding as a "dust cover" in the eustachian tube and entrapping as a "flypaper" against lectin in the transitional area of the middle ear.

Localization of sugar-binding sites in Staphylococcus aureus using gold-labeled neoglycoprotein.

We studied post- and pre-embedding staining of sugar-binding sites on thin sections of Staphylococcus aureus with an electron microscopic neoglycoprotein-gold technique. Although gold particles of cellobiosyl bovine serum albumin (BSA)-glycosylated BSA-, lactosyl BSA-, and melibiosyl BSA-gold did not label, heavy labeling of N-acetylglucosaminide-BSA-gold was observed in both the cell wall and the cytoplasm on Spurr-embedded thin sections of S. aureus. Inhibition of labeling with wheat germ agglutinin-biotin and N-acetylglucosaminidase indicated that the labeling was due to N-acetylglucosamine. These data suggested that molecules that bind specifically with N-acetylglucosamine occur in the cell wall and cytoplasm of S. aureus. Pre-embedding staining revealed that these molecules are abundant at the surface of the cell wall and that the abundance differs depending on the bacterial strain. An N-acetylglucosamine-specific lectin-like substance, glucosaminidase, and toxins are proposed as candidates for molecules responsible for the labeling, and the possible functional significance of the findings is discussed briefly.

Introductory remarks on glycoconjugate histochemistry

In these opening remarks, many typical methods of demonstrating glycoconjugates were presented. Several such classical demonstration methods at the electron microscopic level have been revived to achieve a sufficient electron density, which makes it possible to use these methods routinely at that level. To detect sugar residues at the electron microscopic level, labeled lectins were used by means of preembedding and postembedding staining. Combining these methods, sugar residues in relation to cell organellae have been successfully detected. Many such examples were reported in this presentation.

Immunolocalization of a Drosophila Phosphatidylinositol Transfer Protein (rdgB) in Normal and <italic>rdg</italic>A Mutant Photoreceptor Cells with Special Reference to the Subrhabdomeric Cisternae

Distribution of rdgB protein, which was recently shown to be a Drosophila phosphatidylinositol transfer protein, was studied in the photoreceptor cells of compound eyes of normal and rdgAPC47 mutant of Drosophila melanogaster by immunoelectron microscopy using (1) pre-embedding HRP staining, (2) pre-embedding NANOGOLD labeling followed by siliver enhancement, (3) and post-embedding colloidal gold labeling methods. In normal cells, immunoreactivity was localized to the membranes of subrhabdomeric cisternae (SRC) and adjacent plasma membranes at the bases of photoreceptive microvilli. In rdgAPC47 mutant cells, whose SRC gradually degenerate, the immunoreactivity in the plasma membranes at the bases of microvilli disappeared in parallel with the degeneration of SRC. Instead, the abnormally proliferated membranous structures became labeled by the antibody. These results indicate that the rdgB protein is normally localized to the SRC membranes and probably adjacent photoreceptive membranes, suggesting the involvement of the rdgB protein in the phosphatidylinositol transfer between SRC and photoreceptive membranes.

Fixation and embedding.

AbstracatFor electron microscopic immunocytochemistry, the fixation procedure is always a compromise between good morphological preservation and retention of antigenicity (, ). It is preferable to fix tissues by perfusion, but if that is not possible, the time between removal of the tissue and fixation should be kept as short as possible. The fixative used will depend on whether the immunogold labeling will be done before or after the samples are embedded and on how resistant the antigen is to the fixative. In order to preserve the ultrastructural detail, it is desirable to include glutaraldehyde in the fixative. However, some antigens are extremely sensitive to crosslinking by glutaraldehyde, and the concentration of glutaraldehyde will have to be reduced or, in the most extreme cases, omitted entirely. For pre-embedding staining, it is possible to immunolabel the samples prior to fixation or after only a very mild fixation. Following incubation in the primary antibody or at subsequent steps, the samples can then be refixed in a stronger fixative, such as 2% glutaraldehyde, to give good morphological preservation. For postembedding labeling, it is not possible to refix the tissue after immunolabeling. Therefore, the composition of the initial fixative must be such that morphological detail and antigenicity are both preserved. The composition of the fixative will have to be determined empirically for each antigen.

Pre-embedding labeling methods.

Colloidal gold conjugates generally do not readily penetrate cells, even after permeabilization. Therefore, their use in pre-embedding immunostaining has been largely restricted to labeling cell-surface antigens for scanning or transmission electron microscopy or for tracing endocytic pathways in living cells. One nanometer gold conjugates that do penetrate cells and tissues much more readily have also been used successfully to immunolabel intracellular structures. For pre-embedding labeling, all of the immunostaining is done prior to embedding the tissue in resin or preparing the samples for scanning electron microscopy. This chapter provides methods for pre-embedding staining with unconjugated primary antibody or with primary antibody conjugated to colloidal gold. The use of colloidal gold for tracing endocytic pathways is also given.

Electron microscopic immunocytochemistry of glutamate‐containing nerve fibers in the taste bud of mudpuppy (Necturus maculosus)

The presence of glutamate immunoreactivity (glu-IR) in the nerve fibers of the mudpuppy taste bud was investigated by electron microscopy. Pre-embedding staining with avidin-biotin-peroxidase complex (ABC) and post-embedding staining with 5 nm colloid gold conjugates were used separately to identify immuno-stained structures. We have found the following: 1) the majority of the nerve fibers innervating the mudpuppy taste bud are unmyelinated; 2) about 85% of nerve fibers located at the base of the taste bud and about 60% of the nerve fibers located between the taste cells show glu-IR by pre-embedding staining; 3) there is a preferential staining of the glu-IR in the nerve fibers of the mudpuppy taste bud; and 4) the distribution of the colloidal gold particles in the nerve fibers is 1.5 to 2 times denser than that of the staining in the connective tissue background or cellular profiles of taste cells. From the distribution and pattern of the nerve fibers obtained in the thick and thin sections, we conclude that the mudpuppy taste bud is innervated by glutamate-containing unmyelinated nerve fibers.

Sulfated Glycoconjugates Demonstrated in Combination with High Iron Diamine Thiocarbohydrazide-Silver Proteinate and Silver Acetate Physical Development

Sulfated glycoconjugates in epithelial cells and mesenchymal cells were investigated after staining with high iron diamine-thiocarbohydrazide-silver proteinate. One purpose of the experiment was to apply a new physical developed to the staining. Instead of silver nitrate, silver lactate or silver bromide, we used silver acetate as an ion donor. This new method allowed physical development under normal lighting conditions, and resulted in the reduction of background staining even after amplification. As the developer did not contain gum arabic, troublesome treatment was not necessary. The time required for staining was very short and the electron density of the final reaction product was high and easily identifiable under the electron microscope. Fixing was not necessary. Very small amounts of reactive substance were detectable after physical development. This developmental procedure has been applied to both the preembedding staining and postembedding staining of sulfated glycoconjugates. The results obtained using this method are presented.

Keratin Expression in Merkel Cells of Fetal Rat Skin

The cytokeratin expression of Merkel cells in fetal rat skin was studied by light- and electron microscopy. Employing a pre-embedding staining method, 2 monoclonal anti-keratin antibodies (RCK-102, MA-902) were shown to stain Merkel cells specifically. Neighbouring keratinocytes were unstained. The staining reaction seems to be based on the expression of 52.5 kD cytokeratin.

Antibodies against neuroactive amino acids and neuropeptides. I. A new two-step procedure for their conjugation to carrier proteins and the production of an anti-Met-enkephalin antibody reactive with glutaraldehyde-fixed tissues.

We developed a new two-step procedure to couple haptens to bovine serum albumin (BSA) via glutaraldehyde (GA). After activation of BSA with excess GA and removal of unreacted GA, the hapten was bound to the activated protein in a second step. This two-step procedure is easy to use, the desired molecular ratio of coupled hapten to protein is conveniently adjusted, and no visible precipitation of the conjugate is detected. Using a low peptide concentration, nearly 50% of the inserted haptens are bound to the protein, and unbound expensive peptide can be recovered after Sephadex chromatography. Antisera to neuroactive amino acids (GABA, glycine, and glutamate) and neuropeptides (Met-enkephalin) were prepared by immunization of rabbits with these conjugates. Immunological analysis of immune sera by dot-blot and ELISA techniques and subsequent removal of crossreactivities by solid-phase adsorption yielded monospecific antibodies, which were further purified by affinity chromatography. The immunocytochemical specificities of these purified antibodies were verified in adjacent sections of GA-fixed rat spinal cord. Pre-embedding staining with anti-Met-enkephalin in combination with post-embedding staining for amino acids such as GABA allowed double staining of the two antigens in a single semi-thin section.