Two approaches to double post-embedding immunogold labeling of freeze-substituted tissue embedded in low temperature Lowicryl HM20 resin

Mahendrasingam, Shanthini; C, Wallam; Hackney, Carole M.

doi:10.1016/s1385-299x(03)00040-0

Cited by 24 publications

(13 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Alternatively, freeze-substitution and embedding of fixed brain tissue in Lowicryl HM20 has demonstrated excellent antigen preservation and suitability for immunogold localization of neural antigens in the electron microscope (van Lookeren Campagne et al, 1991). The latter approach, which was used for the immuno-gold detection of FG in the present study, has allowed for the high sensitivity detection of a variety of amino acids, peptides and proteins, including transmitter substances, vesicular transporters, and receptors (Larsson et al, 2001; Mahendrasingam et al, 2003; Persson and Broman, 2004). Thus, FG-labeled neurons and their synaptic inputs may now be studied in the same tissues, allowing for detailed analyses of neuronal circuits.…”

Section: Discussionmentioning

confidence: 99%

Retrogradely transported fluorogold accumulates in lysosomes of neurons and is detectable ultrastructurally using post-embedding immuno-gold methods

Persson

Havton

2009

Journal of Neuroscience Methods

View full text Add to dashboard Cite

For ultrastructural studies, it is of great interest to be able to combine anatomical tracer techniques with sensitive immunohistochemical methods. Fluorogold (FG) is a fluorescent and retrogradely transported anatomical tracer, which is commonly used to label neurons in the brain and spinal cord for light microscopic studies. We here describe a method for detecting FG-labeled somata in the electron microscope using a high resolution post-embedding immuno-gold method. For this purpose, spinal motoneurons were retrogradely labeled by an intraperitoneal injection of FG in the adult rat. The rats were intravascularly perfused with a fixative solution containing 2% paraformaldehyde and 1-2% glutaraldehyde. Vibratome sections of spinal cord tissues were cryo-protected in glycerol, freeze-substituted in methanol containing uranyl acetate, and embedded in the Lowicryl HM20 resin at low temperatures. Electron microscopic analysis demonstrated atypical lysosome-like structures in the cytoplasm of FG-labeled motoneurons. Subsequent post-embedding immuno-gold labeling demonstrated prominent accumulation of FG in numerous lysosomes but not in other organelles or cytoplasmic compartments of the labeled neurons. The protocol is versatile and allows for combining anatomical tracing of neurons with e.g. neuro-transmitter studies in the electron microscope. We suggest that the described method for sensitive detection of FG in the spinal cord may also have broad applicability to other areas of the central nervous system.

show abstract

Section: Discussionmentioning

confidence: 99%

Retrogradely transported fluorogold accumulates in lysosomes of neurons and is detectable ultrastructurally using post-embedding immuno-gold methods

Persson

Havton

2009

Journal of Neuroscience Methods

View full text Add to dashboard Cite

show abstract

“…The IEM approach, particularly associated with nanogold labelling technology, might efficiently contribute to the elucidate of the function of PrP c , thus yielding powerful information for the better understanding of prion diseases (Fournier and Grigoriev 2001). In this direction, a highly promising approach, which consists of the development of a fixation technique combined with acrylate embedment, has shown its efficiency in the detection of two presynaptic proteins by doublelabelling (Mahendrasingam et al 2003).…”

Section: Discussionmentioning

confidence: 99%

Cellular prion protein electron microscopy: attempts/limits and clues to a synaptic trait. Implications in neurodegeneration process

Fournier

2008

Cell Tissue Res

View full text Add to dashboard Cite

Prion diseases are caused by an infectious agent constituted by a rogue protein called prion (PrP Sc) of neuronal origin (PrP c) and are exemplified by Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. Considerable efforts have been made to understand the cerebral damage caused by these diseases but a clear comprehensive view cannot be achieved without defining the neurophysiological function of PrP c. This lack of information is in part attributable to our ignorance of the precise localization of PrP c in the brain neuronal cell. One relevant option to explore this aspect is to undertake PrP immunohistochemistry at the electron-microscopy level, knowing that this challenge raises major technical constraints. In describing the attempts and restrictions of the various approaches used, we review here the efforts that have been invested in this particular field of prionology. The common result emerging from these contributions is that the synapse could be the site at which PrP c exerts its critical activity. This location suggests, in the perspective of synaptic regulation, that PrP c can be assigned multiple biological functions and supports the novel concept that prion-like changes are involved in long-term memory formation. The synaptic trait of PrP c and PrP Sc suggests that synapse loss is the key event in neuronal death. Interestingly, synaptic alterations are also considered to be predominant in the pathophysiological mechanism in Alzheimer, Parkinson and Huntington diseases. All these brain disorders, characterized by the formation of a specific amyloid protein of synaptic origin, can be classified under the heading of amyloidogenic synaptopathies.

show abstract

“…The samples were blocked and incubated with single primary antibodies (anti‐EhCpn60, anti‐TvRbr, anti‐EhNifS or anti‐EhNifU; all 1:50 and 1:200 dilutions – pre‐immune serum or blocking solution were used in control grids) followed with secondary goat anti‐rabbit IgG conjugated to immunogold particles of either 10 nm or 20 nm in diameter (BB International; 1:100 dilution in blocking buffer). Double labeling experiments were carried out in the same way but with an additional paraformaldehyde vapor treatment step between antibody incubations as previously described (Mahendrasingam et al. , 2003).…”

Section: Methodsmentioning

confidence: 99%

Bacterial-type oxygen detoxification and iron-sulfur cluster assembly in amoebal relict mitochondria

Maralikova

Ali

Nakada‐Tsukui

et al. 2010

Cellular Microbiology

View full text Add to dashboard Cite

SummaryThe assembly of vital reactive iron-sulfur (Fe-S) cofactors in eukaryotes is mediated by proteins inherited from the original mitochondrial endosymbiont. Uniquely among eukaryotes, however, Entamoeba and Mastigamoeba lack such mitochondrial-type Fe-S cluster assembly proteins and possess instead an analogous bacterial-type system acquired by lateral gene transfer. Here we demonstrate, using immunomicroscopy and biochemical methods, that beyond their predicted cytosolic distribution the bacterial-type Fe-S cluster assembly proteins NifS and NifU have been recruited to function within the relict mitochondrial organelles (mitosomes) of Entamoeba histolytica. Both Nif proteins are 10-fold more concentrated within mitosomes compared with their cytosolic distribution suggesting that active Fe-S protein maturation occurs in these organelles. Quantitative immunoelectron microscopy showed that amoebal mitosomes are minute but highly abundant cellular structures that occupy up to 2% of the total cell volume. In addition, protein colocalization studies allowed identification of the amoebal hydroperoxide detoxification enzyme rubrerythrin as a mitosomal protein. This protein contains functional Fe-S centres and exhibits peroxidase activity in vitro. Our findings demonstrate the role of analogous protein replacement in mitochondrial organelle evolution and suggest that the relict mitochondrial organelles of Entamoeba are important sites of metabolic activity that function in Fe-S proteinmediated oxygen detoxification.

show abstract

Two approaches to double post-embedding immunogold labeling of freeze-substituted tissue embedded in low temperature Lowicryl HM20 resin

Cited by 24 publications

References 11 publications

Retrogradely transported fluorogold accumulates in lysosomes of neurons and is detectable ultrastructurally using post-embedding immuno-gold methods

Retrogradely transported fluorogold accumulates in lysosomes of neurons and is detectable ultrastructurally using post-embedding immuno-gold methods

Cellular prion protein electron microscopy: attempts/limits and clues to a synaptic trait. Implications in neurodegeneration process

Bacterial-type oxygen detoxification and iron-sulfur cluster assembly in amoebal relict mitochondria

Contact Info

Product

Resources

About