scholarly journals A serial multiplex immunogold labeling method for identifying peptidergic neurons in connectomes

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Réza Shahidi ◽  
Elizabeth A Williams ◽  
Markus Conzelmann ◽  
Albina Asadulina ◽  
Csaba Verasztó ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Immunogold Labeling ◽  
Synaptic Connections ◽  
Peptidergic Neurons ◽  
Pigment Dispersing Factor ◽  
Transmission Electron ◽  
Nuchal Organs ◽  
Labeling Method ◽  
High Immunogenicity ◽  
Full Body

Electron microscopy-based connectomics aims to comprehensively map synaptic connections in neural tissue. However, current approaches are limited in their capacity to directly assign molecular identities to neurons. Here, we use serial multiplex immunogold labeling (siGOLD) and serial-section transmission electron microscopy (ssTEM) to identify multiple peptidergic neurons in a connectome. The high immunogenicity of neuropeptides and their broad distribution along axons, allowed us to identify distinct neurons by immunolabeling small subsets of sections within larger series. We demonstrate the scalability of siGOLD by using 11 neuropeptide antibodies on a full-body larval ssTEM dataset of the annelid Platynereis. We also reconstruct a peptidergic circuitry comprising the sensory nuchal organs, found by siGOLD to express pigment-dispersing factor, a circadian neuropeptide. Our approach enables the direct overlaying of chemical neuromodulatory maps onto synaptic connectomic maps in the study of nervous systems.

scholarly journals A Serial Multiplex Immunogold Labeling Method for Identifying Peptidergic Neurons in Connectomes

2015 ◽  
Author(s):  
Reza Shahidi ◽  
Elizabeth Williams ◽  
Markus Conzelmann ◽  
Albina Asadulina ◽  
Csaba Veraszto ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Immunogold Labeling ◽  
Synaptic Connections ◽  
Peptidergic Neurons ◽  
Pigment Dispersing Factor ◽  
Transmission Electron ◽  
Nuchal Organs ◽  
Labeling Method ◽  
High Immunogenicity ◽  
Full Body

Electron microscopy-based connectomics aims to comprehensively map synaptic connections in neural tissue. However, current approaches are limited in their capacity to directly assign molecular identities to neurons. Here, we use serial multiplex immunogold labeling (siGOLD) and serial-section transmission electron microscopy (ssTEM) to identify multiple peptidergic neurons in a connectome. The high immunogenicity of neuropeptides and their broad distribution along axons, allowed us to identify distinct neurons by immunolabeling small subsets of sections within larger series. We demonstrate the scalability of siGOLD by using 11 neuropeptide antibodies on a full-body larval ssTEM dataset of the annelid Platynereis. We also reconstruct a peptidergic circuitry comprising the sensory nuchal organs, found by siGOLD to express pigment-dispersing factor, a circadian neuropeptide. Our approach enables the direct overlaying of chemical neuromodulatory maps onto synaptic connectomic maps in the study of nervous systems.

Immunogold labeling of Pneumocystis carinii for Electron microscopy

1991 ◽  
Vol 49 ◽  
pp. 270-271
Author(s):  
Michael P. Goheen ◽  
Marilyn S. Bartlett ◽  
James W. Smith
Keyword(s):  
Electron Microscopy ◽  
Pneumocystis Carinii ◽  
Severe Pneumonia ◽  
Culture Fluid ◽  
Immunogold Labeling ◽  
Antigenic Sites ◽  
Transmission Electron ◽  
Response To Chemotherapy ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome

Studies of the biology of Pneumocystis carinii (PC) are of increasing importance because this extracellular pathogen is a frequent source of severe pneumonia in patients with acquired immunodeficiency syndrome (AIDS) and is a leading cause of mortality in these patients. Immunoelectron microscopic localization of antigenic sites on the surface of PC would improve the understanding of these sites and their role in pathenogenisis of the disease and response to chemotherapy. The purpose of this study was to develop a methodology for visualizing immunoreactive sites on PC with transmission electron microscopy (TEM) using immunogold labeled probes.Trophozoites of PC were added to spinner flask cultures and allowed to grow for 7 days, then aliquots of tissue culture fluid were centrifuged at 12,000 RPM for 30 sec. Pellets of organisims were fixed in either 1% glutaraldehyde, 0.1% glutaraldehyde-4% paraformaldehyde, or 4% paraformaldehyde for 4h. All fixatives were buffered with 0.1M Na cacodylate and the pH adjusted to 7.1. After fixation the pellets were rinsed in 0.1M Na cacodylate (3X), dehydrated with ethanol, and immersed in a 1:1 mixture of 95% ethanol and LR White resin.

Light Microscopy, Confocal Laser Scanning Microscopy And Scanning And Transmission Electron Microscopy Of Cerebellar Golgi Cells.

1999 ◽  
Vol 5 (S2) ◽  
pp. 1302-1303
Author(s):  
O. Castejόn ◽  
P Sims
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Synaptic Connections ◽  
Teleost Fishes ◽  
Golgi Cells ◽  
Transmission Electron ◽  
Molecular Layers

The cerebellar cortex of albino mice, hamsters, teleost fishes, primates and human have been examined by correlative microscopy to study the Golgi cell soma, dendritic processes, axonal plexus and synaptic connections in the granular and molecular layers. For light microscopy (LM) toluidinc blue stained-plastic embedded scmithin sections and Golgi light microscopy preparations were used. For confocal laser scanning microscopy (CLSM) of hamster cerebellum the FM4-64 fluorescent stain was used as intracellular tracer (1). Conventional and high resolution scanning electron microscopy (SEM) of teleost fishes, primates and human were coated with gold-palladium and chromium (2). I Transmission electron microscopy (TEM). either by ullrathin sections or frccze-clching replicas, were examined to characterize synaptic connections in the granular and molecular layers. The Golgi cells appeared in the granular layer as polygonal, stellate, round or fusiform microncurons. 10-25 μm in maximal dimension, surrounded by the granule cell groups. Golgi light microscopy.

An Innovative Triple Immunogold Labeling Method to Investigate the Hemopoietic Stem Cell NicheIn Situ

2009 ◽  
Vol 15 (5) ◽  
pp. 403-414 ◽  
Author(s):  
Sarah L. Ellis ◽  
Brenda Williams ◽  
Stephen Asquith ◽  
Ivan Bertoncello ◽  
Susan K. Nilsson
Keyword(s):  
Stem Cell ◽  
Immunogold Labeling ◽  
Hemopoietic Stem Cell ◽  
Hemopoietic Stem Cells ◽  
Innovative Method ◽  
Transmission Electron ◽  
Rare Cells ◽  
Cellular Phenotypes ◽  
Labeling Method

AbstractThe ultrastructural study of rare cells within their nichein situis very difficult. We have developed a method for locating individual transplanted cells and simultaneously identifying and analyzing the molecules and cellular phenotypes surrounding themin situusing transmission electron microscopy. This innovative method involves triple immunogold labeling combined with serial ultrathin sectioning. We demonstrate the validity of this approach by examining the niche of individual transplanted cells from a population highly enriched for hemopoietic stem cells and the ultrastructural expression of two key stem cell regulatory molecules, hyaluronic acid and osteopontin. In addition, we describe the phenotypes of the surrounding cells.

scholarly journals Optimization of Immunogold Labeling TEM

2002 ◽  
Vol 50 (6) ◽  
pp. 863-873 ◽  
Author(s):  
Ramandeep Kaur ◽  
Kanak L. Dikshit ◽  
Manoj Raje
Keyword(s):  
Electron Microscopy ◽  
Horse Serum ◽  
Skim Milk ◽  
Blocking Agent ◽  
Immunogold Labeling ◽  
Dot Blot ◽  
Blocking Agents ◽  
Transmission Electron ◽  
Assay Procedure ◽  
Accurate Picture

We developed an ELISA-based method for rapid selection of optimal blocking agents to be used in antigen quantification by immunogold labeling electron microscopy. Casein, skim milk, BSA from two sources, acetylated BSA, fish skin gelatin, horse serum, and goat serum were tested for their ability to block nonspecific binding of antibody to recombinant Vitreoscilla hemoglobin (VHb) antigen expressed in Escherichia coli cells by ELISA and the results were confirmed by quantitative immunogold labeling transmission electron microscopy (TEM). Ability to minimize NSB was also evaluated by dot-blot and Western blotting methods. The results demonstrated that ELISA was most accurate in predicting the most efficient blocking agent for TEM. Existing methods could not provide an accurate picture of the ability of various reagents to suppress background labeling. The sensitivity of detection of antigens by immunoelectron microscopy depends on the assay procedure being optimized to obtain the highest possible signal along with as low a background (noise) as possible. Our study indicated that an ELISA-based evaluation of various blocking agents could help in the rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigens by TEM.

Immunogold labeling of HTLV-III/LAV in H9 cells studied by transmission electron microscopy

1986 ◽  
Vol 13 (3) ◽  
pp. 265-269 ◽  
Author(s):  
D. Pekovic ◽  
S. Garzon ◽  
H. Strykowski ◽  
D. Ajdukovic ◽  
M. Gornitsky ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Immunogold Labeling ◽  
Transmission Electron

scholarly journals Pitfalls of immunogold labeling: analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy.

1987 ◽  
Vol 35 (8) ◽  
pp. 843-853 ◽  
Author(s):  
G B Birrell ◽  
K K Hedberg ◽  
O H Griffith
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Light Microscopy ◽  
Tannic Acid ◽  
Cultured Cells ◽  
Immunogold Labeling ◽  
Fish Gelatin ◽  
Nonspecific Binding ◽  
Gold Particles ◽  
Transmission Electron

The immunogold method is widely used to localize, identify, and distinguish cellular antigens. There are, however, some pitfalls that can lead to nonspecific binding, particularly in cytoskeletal studies with gold probes prepared from small gold particles. We present a list of suggestions for minimizing nonspecific binding, with particular attention to two problems identified in this study. First, we find that the method used to prepare the colloidal gold particles affects the degree of nonspecific binding. Second, the standard BSA-stabilized small gold probes evidently possess exposed regions that bind to the proteins of cytoskeletal preparations. This was investigated in whole-mount cytoskeletal preparations of cultured cells by use of light microscopy, transmission electron microscopy, and photoelectron microscopy of silver-enhanced specimens. Gold probes were made from approximately 5-nm particles generated by reduction of HAuCl4 with three different reducing agents: white phosphorus, sodium borohydride, and citrate-tannic acid. All three preparations stabilized in the conventional way showed significant levels of nonspecific binding, which was highest with citrate-tannic acid. This problem was largely solved with all three types of probes by including fish gelatin in the probe buffer, by substituting fish gelatin for the BSA stabilizer used to prepare the probes, or by pre-adsorption methods. Application of these techniques resulted in clear immunogold labeling patterns with minimal nonspecific background.

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