eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Tissue Preparation
<ol>
	<li>Prepare 4% formaldehyde in 0.1 M phosphate buffer pH 7.4 on the day before perfusion. Store at 4 &#176;C.</li>
	<li>Transcardially perfuse the deeply anesthetized mice (3.5% isoflurane) via the left ventricle with 10 ml ice-cold phosphate-buffered saline (PBS), then with 40 ml ice-cold formaldehyde (f...

an immunofluorescence staining technique to detect adult hippocampal neurogenesis

Source: Ansorg, A., et al. <a href="https://app.jove.com/v/52551">Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis.</a> J. Vis. Exp. (2015)
The video illustrates an immunofluorescence staining method used to examine neurogenesis in the adult hippocampus. Initially, a mouse brain section undergoes treatment with primary antibodies directed at marker proteins associated with neurogenesis in neuronal progenitor cells. Next, it is labeled with secondary antibodies tagged with fluorophores. The brain section is then observed under a confocal microscope to distinguish various subtypes of progenitor cells, based on the expression patterns of distinct combinations of marker proteins.

<img alt="Figure 1" src="/files/ftp_upload/22108/22108_Figure1.jpg" /> 
Figure 1. Schematic illustration of transferring microtome slices into a 24-well plate. Start at A1 and place subsequent slices into row A; after A6, go to the next row B, and so forth. When reaching D6, go back to A1 and continue. This arrangement of slices allows for quantification of every nth section of an entire brain. For quantification of newborn cells, take every 6th...

An Immunofluorescence Staining Technique to Detect Adult Hippocampal Neurogenesis

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Tissue ...

Take a murine brain section consisting of the hippocampal dentate gyrus, a site for neurogenesis composed of neuronal progenitors at distinct differentiation stages.
Transfer the section into a permeabilization-blocking solution. Detergent molecules in the solution permeabilize the cells, while the proteins block the non-specific binding sites.
Add a primary antibody cocktail targeting specific neurogenesis markers, a microtubule-associated protein, and an intermediate filament protein.
Remove the unbound antibodies. Introduce different fluorophore-labeled secondary antibodies binding to the primary antibodies.
Add serum proteins from the same host as one of the primary antibodies, saturating the paratopes on the secondary antibodies.
Introduce monovalent Fab fragments that saturate the serum proteins&#39; epitopes, preventing non-specific binding.
Add a second same-host-raised primary antibody, binding to another intermediate filament protein.
Introduce fluorophore-labeled secondary antibodies targeting the second primary antibody.
Using fluorescence microscopy, identify the neuronal progenitor subtypes expressing unique marker combinations.

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Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

JoVE Journal

Developmental Biology

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

Neuroscience

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to obtain commercial monoclonal or polyclonal antibodies that recognize specific cell structures and proteins in parasites. Besides, there are few commercial antibodies available to label trypanosomatids. Usually, polyclonal antibodies against parasites are prepared in-house and could be more challenging to use in combination with other antibodies produced in the same species. Here, the protocol demonstrates how to use polyclonal and monoclonal antibodies raised in the same species to perform double labeling immunofluorescence to study host cell and pathogen interactions. To achieve the double labeling immunofluorescence, it is crucial to incubate first the mouse polyclonal antibody and then follow the incubation with the secondary mouse IgG antibody conjugated to any fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the primary antibody from being recognized by the next secondary antibody. Then, a mouse monoclonal antibody and its specific IgG subclass secondary antibody conjugated to a different fluorochrome are added to the sample at the appropriate times. Additionally, it is possible to perform triple labeling immunofluorescence using a third antibody raised in a different species. Also, structures such as nuclei and actin can be stained subsequently with their specific compounds or labels. Thus, these approaches presented here can be adjusted for any cell whose sources of primary antibodies are limited.

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to ...

An Immunofluorescence Staining Technique to Detect Adult Hippocampal Neurogenesis

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