Pre-embedding Immunoelectron Microscopy Research Articles

An immuno-electron microscopic study on interactions among dendritic cells, macrophages and lymphocytes in the human palatine tonsil.

The sites of interaction between antigen-presenting cells (dendritic cells, macrophages) and lymphocytes in the human palatine tonsil were investigated by pre-embedding immuno-electron microscopy and acid-phosphatase histochemistry. Used in this study were: an S-100 protein antiserum recognizing Langerhans cells and interdigitating cells, OKT6 monoclonal antibody reacting with surface antigens of Langerhans cells, and Leu-3a monoclonal antibody directed to surface antigens of helper-T-cells. In the stratified squamous epithelium, Langerhans cells with characteristic rod-shaped or racket-shaped Birbeck granules exhibited S-100 protein and OKT6 immunoreactivites, and frequently extended long processes to adjacent lymphocytes. The subepithelial area contained a significant number of macrophages, some of which were closely apposed to interdigitating cells with S-100 protein immunoreactivity. Occasionally, macrophages with acid-phosphatase-positive phagosomes and/or lysosomes extended short processes to neighboring lymphocytes. In the interfollicular area, some interdigitating cells and a few Langerhans cells were seen in proximity to or in contact with lymphocytes stained with the Leu-3a antibody. These findings support previous in vitro studies suggesting the induction of T-cell activation by antigen-presenting cells. They further indicate that T-cell activation by the individual antigen-presenting cells takes place in different areas of the human palatine tonsil through a variety of cell-to-cell interactions.

Simultaneous Colloidal Gold Immunoelectronmicroscopy Labeling of CD1a, HLA-DR, and CD4 Surface Antigens of Human Epidermal Langerhans Cells

The simultaneous demonstration of three surface antigens of Langerhans cells (LC) within LC-enriched fresh epidermal cell suspensions from normal human skin was achieved, by means of a triple immunogold (IG) staining, using commercially available monoclonal antibodies (moAb) and immunoreagents, in a simple pre-embedding immunoelectronmicroscopy (IEM) procedure. As a result, suspended LC were triple-stained as follows: gold particles of 40 nm revealed the CD1 a antigen; gold particles of 20 nm revealed the HLA-DR antigen; and gold particles of 5 nm revealed the CD4 antigen. All the observed epidermal Birbeck granule-bearing LC were triple IG stained, thus simultaneously expressing the three surface differentiation antigens, which are therefore different from but coexisting with each other. The present investigation assesses the constant simultaneous expression by Birbeck granules bearing LC of not only CD1a and HLA-DR antigens, but also CD4 antigen. The occurrence is therefore excluded of both CD1a-positive HLA-DR-negative LC subpopulation and CD4-negative LC subpopulation, presumably due to the different sensitivity of the various procedures performed. The hypothetical occurrence of CD4-positive, CD1a-, and/or HLA-DR-negative LC subpopulations is ruled out. This study reaffirms indeed the high specificity and sensitivity of the IG-IEM method for a precise detection of the cell surface antigens of LC, and states the suitability of the IG labeling even for accurate multiple IEM stainings of LC.

Fine structures of nerve fibers with corticotropin-releasing factor-like immunoreactivity in the rat lateral septum

The fine structures of nerve fibers with corticotropin-releasing factor (CRF)-like immunoreactivity in the rat lateral septum were investigated by means of preembedding immunoelectron microscopy. A number of CRF axon terminals formed synapses with cell bodies of non-immunoreactive septal neurons. They occasionally had broad terminal bulges whose subregions showed little or no immunoreactivity for CRF. CRF axon terminals were also in synaptic contact with non-immunoreactive dendrites or dendritic spines. Some dendrites with CRF were postsynaptic to non-immunoreactive axon terminals.

Comparative immunoelectron microscopy with monoclonal antibodies on yellow fever virus-infected cells: Pre-embedding labelling versus immuno-cryoultramicrotomy

To study the intra- and extracellular distribution of yellow fever virus 17D (YFV)-specific antigens, pre-embedding immunoelectron microscopy (IEM) and IEM on ultrathin frozen sections were carried out comparatively using monoclonal antibodies (MAB) and YFV-infected cells. In addition, three electrondense marker systems (IgG-ferritin and IgG-gold and protein A-gold) were compared for their efficiency in detecting bound MAB. Pre-embedding immuno-label ling was performed in microtest plates followed by in situ embedding and immunocryoultramicrotomy was performed using pellets of sucrose-infused cells. In both procedures, cells were prefixed with different concentrations of glutaraldehyde (GA). In pre-embedding IEM virus-specific antigens could be detected on the envelopes of extracellular virions with YFV-neutralizing MAB. Using immunocryoultramicrotomy, neutralizing MAB bound to intracellular mature virions as well as to viral antigens incorporated into cytoplasmic membranes. A concentration of 1% GA destroyed antigenicity entirely, while with 0.25% and 0.1% GA immunoreactivity was retained for more than 3 mth. Some highly reactive MAB labelled antigen significantly in pre-embedding IEM, when used at concentrations of 1 ng/ml. Immunocryoultramicrotomy was 10–100 times less sensitive. On cryosections colloidal gold was the marker of choice, due to the fact that it showed less nonspecific sticking to intracellular components and that it was easily detectable on highly contrasted cryosections. Owing to their higher sensitivity, IgG-ferritin conjugates were preferred in pre-embedding IEM.

Immunocytochemical localization of catalase in rat liver.

The intracellular localization of catalase has been studied using monospecific Fab fragments against rat liver catalase (RLC) and preembedding immunoelectron microscopy. Purified RLC, exhibiting a single band on sodium dodecyl sulfate gel electrophoresis, was used for the immunization of rabbits. The anti-RLC IgG was purified by affinity chromatography. Fab fragments were obtained by papain digestion and were labeled with horseradish peroxidase (HRP) using a modified two-step procedure and glutaraldehyde as coupling agents. Livers were perfused with 4% depolymerized paraformaldehyde and chopper sections were incubated with HRP-labeled Fab fragments against RLC. Because of the limited penetration of labeled Fab fragments into chopper sections a simple method for preparation of a cell suspension from aldehyde-fixed livers was devised. Adequate staining of more than 90% of cell was obtained by incubation of cell suspensions for 12--18 hr with the labeled antibody. By light microscopy specific staining was present in fine granules in the cytoplasm of hepatocytes. By electron microscopy the electron-dense reaction product was localized in the matrix of peroxisomes with no reaction in the endoplasmic reticulum and the Golgi complex. In some hepatocytes, positively reacted peroxisomes were seen side by side with unstained particles. Although focal diffusion was noted around a few peroxisomes, no evidence of cytoplasmic catalase independent of peroxisomes was found. These observations indicate that in rat liver peroxisomes are the only organelle containing substantial amounts of catalase antigen and rule out any involvement of the endoplasmic reticulum and the Golgi complex in the sequestration of this protein.

Pre-embedding immunoelectron microscopy by peroxidase-labeled antibody method - Applications to endocrinology.

Pre-embedding immunoelectron microscopy has demonstrated the ultra-structural localization of antigens in various intracellular organelles, including cisternae of rough endoplasmic reticula, pen-nuclear spaces, Golgi saccules, cytosol, secretory granules and mitochondria.In this report, major staining procedures of this method are briefly described and its applications to endocrinology are demonstrated.

Polyethylene glycol embedded tissue sections for immunoelectronmicroscopy.

Several methods for tissue embedding in polyethylene glycol (PEG) were compared with regard to their applicability for pre-embedding immunoelectronmicroscopy. Existing embedding procedures gave unsatisfactory results and therefore a modified procedure was developed. This method, consisting of very brief tissue infiltration with PEG 1500, to which 3% water is added, allowed adequate tissue sectioning. Using these sections for preembedding immunoelectronmicroscopical localisation of glucagon in bovine pancreatic islets adequate ultrastructural morphology was obtained in combination with excellent preservation of peptide hormone immunoreactivity.