Mouse Neo-Cortex
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Title: Mouse Neo-Cortex
Description: Mouse neo-cortex with endogenous yellow fluorescent protein used to visualize neuronal cell bodies, axons, dendrites and dendritic spines. The longer red structures are axon initial segments and the smaller red/green structures are the nodes of Ranvier (red) flanked by the para-nodes (green). Scale bar 10 µm.
An eight week old male C57bl/6 mouse was exposed to traumatic brain injury and then transcardially perfused with heparinized saline followed by 4% paraformaldehyde. The brain was vibratome sectioned at 40 µm and stained free-floating before mounting.
The image consists of three colors:
Yellow is endogenous expression of yellow fluorescent protein (YFP) in a subset of neurons.
Red is immunohistochemically labeled AnkyrinG.
Green is immunohistochemically labeled Caspr.
The following antibodies were used:
Primary A: Anti-AnkyrinG, mouse IgG2a (NeuroMab)
Primary B: Anti-Caspr, mosue IgG1 (NeuroMab)
Secondary A: Anti mouse-IgG2a, AlexaFluor-594 (Molecular Probes)
Secondary B: Anti mouse-IgG1, AlexaFluor-488 (Molecular Probes)
Challenges tackled:
Since both primary antibodies are made in mouse the secondaries had to be iso-type specific. The specificity of the secondaries were confirmed by performing two single stains with the secondaries reversed (Primary A + Secondary B and in a separate stain Primary B + Secondary A). This did not result in any specific staining pattern.
The tissue was not post-fixed since this was found to almost completely eliminate the AnkyrinG signal.
Cold water fish skin gelatin was used to block unspecific antibody binding since normal goat serum resulted in an unacceptably high background signal when staining for Caspr.
The expression of YFP is much higher in the neuronal cell bodies compared to the neurites (axons and dendrites). To allow simultaneous imaging without over- or under-exposing either one the image was acquired with the neurites deliberately underexposed and later visualized by adjusting the gamma-value of the acquired image.
To be able to determine if trauma-induced axonal injuries primarily occur in certain axonal subdomains these first have to be labeled using markers such as AnkyrinG and Caspr.
Anders Hånell, Staff, Anatomy and Neurobiology
The images were acquired using a Zeiss LSM710 system with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. Each color was acquired sequentially to reduce the risk of bleed-through between the channels. The absence of bleed-through could be confirmed since the fluorophores are spatially separated. The image was acquired as a confocal z-stack with 14 slices and is displayed as a maximum intensity projection. The gamma-value was adjusted for yellow channel but the image is otherwise unaltered.
An eight week old male C57bl/6 mouse was exposed to traumatic brain injury and then transcardially perfused with heparinized saline followed by 4% paraformaldehyde. The brain was vibratome sectioned at 40 µm and stained free-floating before mounting.
The image consists of three colors:
Yellow is endogenous expression of yellow fluorescent protein (YFP) in a subset of neurons.
Red is immunohistochemically labeled AnkyrinG.
Green is immunohistochemically labeled Caspr.
The following antibodies were used:
Primary A: Anti-AnkyrinG, mouse IgG2a (NeuroMab)
Primary B: Anti-Caspr, mosue IgG1 (NeuroMab)
Secondary A: Anti mouse-IgG2a, AlexaFluor-594 (Molecular Probes)
Secondary B: Anti mouse-IgG1, AlexaFluor-488 (Molecular Probes)
Challenges tackled:
Since both primary antibodies are made in mouse the secondaries had to be iso-type specific. The specificity of the secondaries were confirmed by performing two single stains with the secondaries reversed (Primary A + Secondary B and in a separate stain Primary B + Secondary A). This did not result in any specific staining pattern.
The tissue was not post-fixed since this was found to almost completely eliminate the AnkyrinG signal.
Cold water fish skin gelatin was used to block unspecific antibody binding since normal goat serum resulted in an unacceptably high background signal when staining for Caspr.
The expression of YFP is much higher in the neuronal cell bodies compared to the neurites (axons and dendrites). To allow simultaneous imaging without over- or under-exposing either one the image was acquired with the neurites deliberately underexposed and later visualized by adjusting the gamma-value of the acquired image.
To be able to determine if trauma-induced axonal injuries primarily occur in certain axonal subdomains these first have to be labeled using markers such as AnkyrinG and Caspr.
Anders Hånell, Staff, Anatomy and Neurobiology
The images were acquired using a Zeiss LSM710 system with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. Each color was acquired sequentially to reduce the risk of bleed-through between the channels. The absence of bleed-through could be confirmed since the fluorophores are spatially separated. The image was acquired as a confocal z-stack with 14 slices and is displayed as a maximum intensity projection. The gamma-value was adjusted for yellow channel but the image is otherwise unaltered.
Identifier: ttlg-05
Citation: “Mouse Neo-Cortex,” VCU Libraries Gallery, accessed May 4, 2024, https://gallery.library.vcu.edu/items/show/2732.