histochemical imaging of cortical modules in flattened brain sections

Histochemical Imaging of Cortical Modules in Flattened Brain Sections

Wash the section tissue in HEPES buffer for 15 minutes with gentle agitation. During the wash, prepare fresh cytochrome oxidase staining solution, and add the DAB component just before using it. Now, incubate the sections in the freshly made staining solution at room temperature with shaker. And keep watch. Depending on the amount of fixation, staining could be observable in 10 minutes, or it may take several hours.If the reaction is going too slow, increase the temperature to 37 degrees Celsius. To stop the reaction, transfer the sections to a bath of 4% PFA, and let them incubate for 15 minutes with gentle agitation. Then, wash the sections three times with HEPES buffer for 10 minutes per wash.Now, mount and dry the sections on glass slides, and dehydrate them using progressively stronger ethanol baths. Then, wash the slides in isopropanol for 5 minutes, followed by xylene for five minutes. After the xylene bath, immediately add a quick hardening non-water based mounting medium. Water-based mediums will degrade the stain. After adding a coverslip, store the slides at 4 degrees Celsius until imaged.

histochemical-imaging-of-cortical-modules-in-flattened-brain-sections

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Synaptic & Cellular Imaging

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<figure class='table'><table class='ck-table-resized'><colgroup><col style='width:33.33%;'><col style='width:33.33%;'><col style='width:33.34%;'></colgroup><tbody><tr><td>Cytochrome oxidase staining</td><td>&#160;</td><td>&#160;</td></tr><tr><td>Cytochrome c from equine heart</td><td>Sigma-Aldrich</td><td>C2506</td></tr><tr><td>3,3'Diaminobenzidine tetrahydrochloride hydrate</td><td>Sigma-Aldrich</td><td>D5637</td></tr><tr><td>HEPES</td><td>Carl Roth</td><td>9105.4</td></tr><tr><td>Mounting or freezing media</td><td>&#160;</td><td>&#160;</td></tr><tr><td>Eukitt (histochemistry)</td><td>Sigma-Aldrich</td><td>3989</td></tr><tr><td>Alcohol dehydration</td><td>&#160;</td><td>&#160;</td></tr><tr><td>Ethanol 100%</td><td>Carl Roth</td><td>9065.3</td></tr><tr><td>Ethanol 96%</td><td>Carl Roth</td><td>P075.3</td></tr><tr><td>2-Propanol</td><td>Carl Roth</td><td>6752.4</td></tr><tr><td>Xylene substitute</td><td>Fluka</td><td>78475</td></tr><tr><td>Devices/tools</td><td>&#160;</td><td>&#160;</td></tr><tr><td>Dumont 55 Forceps - Inox</td><td>Fine Science Tools</td><td>11255-20</td></tr><tr><td>Dumont 5 Forceps - Inox Biology Tip</td><td>Fine Science Tools</td><td>11252-30</td></tr><tr><td>Dumont 5SF Forceps - Inox Super Fine Tip</td><td>Fine Science Tools</td><td>11252-00</td></tr></tbody></table></figure>

Source:&#160;Lauer, S. M.,&#160;et al.&#160;Visualization of Cortical Modules in Flattened Mammalian Cortices.&#160;J. Vis. Exp.&#160;(2018)&#160;This ...

Start with pre-fixed tangential sections of the flattened brain cortical sections. Wash to remove fixative traces.&#160;Stain the sections with a solution of diaminobenzidine or DAB and nickel-ammonium sulfate.&#160;DAB diffuses into cortical neurons and reacts with cytochrome oxidase in mitochondria, forming a dark precipitate. Nickel-ammonium sulfate darkens the precipitate, enhancing contrast.Add paraformaldehyde to stop the staining reaction, then rinse. &#160;Mount the sections on a slide and dehydrate them with ethanol washes. &#160;Rinse with isopropanol and xylene to clear the section and improve light transmission.Apply a mounting medium and put a coverslip on it.&#160;When imaged under a bright-field microscope, within the cortical neurons, the precipitates absorb and scatter light, making them appear dark.In contrast, unstained, cleared areas transmit light and appear bright.This contrast highlights cortical modules as dark, patchy structures against a lighter background, representing active neural clusters involved in processing sensory information.

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Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation.
We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P)+ and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase&#39;s activity. The formazan&#39;s absorbance is therefore a direct measure of the dehydrogenase&#39;s activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays.
Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Here, we present a protocol that can be used to microscopically visualize and quantify activity of dehydrogenases in cells and tissues and its kinetics, function and subcellular localization.

Metabolische Mapping: Quantitative Enzym Cytochemistry und Histochemie die Aktivität des Dehydrogenases in Zellen und Geweben zu bestimmen

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells ...

Forschung

JoVE Journal

Immunologie und Infektion

Abbiategrasso Brain Bank Protocol for Collecting, Processing and Characterizing Aging Brains

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find preventive and curative measures. The most effective way to achieve this is through direct examination of diseased and healthy brain tissue. The authors present a protocol to obtain, process, characterize and store good quality brain tissue donated by individuals registered in an antemortem brain donation program. The donation program includes a face-to-face empathic approach to people, a collection of complementary clinical, biological, social and lifestyle information and serial multi-dimensional assessments over time to track individual trajectories of normal aging and cognitive decline. Since many neurological diseases are asymmetrical, our brain bank offers a unique protocol for slicing fresh specimens. Brain sections of both hemispheres are alternately frozen (at -80 &#176;C) or fixed in formalin; a fixed slice on one hemisphere corresponds to a frozen one on the other hemisphere. With this approach, a complete histological characterization of all frozen material can be obtained, and omics studies can be performed on histologically well-defined tissues from both hemispheres thus offering a more complete assessment of neurodegenerative disease mechanisms. Correct and definite diagnosis of these diseases can only be achieved by combining the clinical syndrome with the neuropathological evaluation, which often adds important etiological clues necessary to interpret the pathogenesis. This method can be time consuming, expensive and limited as it only covers a limited geographical area. Regardless of its limitations, the high degree of characterization it provides can be rewarding. Our ultimate goal is to establish the first Italian Brain Bank, all the while emphasizing the importance of neuropathologically verified epidemiological studies.

This protocol describes a method to track individual brain-aging trajectories through a brain donation program and proper characterization of brains. Brain donors are involved in a long-term longitudinal study including serial multi-dimensional assessments. The protocol contains a detailed description of brain processing and an accurate diagnostic methodology.

Abbiategrasso Brain Bank Protocol zum Sammeln, Verarbeiten und Charakterisieren alternder Gehirne

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find ...

Neurowissenschaften

Histological-Based Stainings Using Free-Floating Tissue Sections

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are widely used to handle the tissue sections during the staining procedure, one approach consists of mounting the sections directly on glass slides, while a second approach, the free-floating, allows for fixed sections to be maintained and stained while suspended in solution. Although slide-mounted and free-floating approaches may yield similar results, the free-floating technique allows for better antibody penetration and thus should be the method of choice when thicker sections are to be used for 3D reconstruction of the tissues, for example when the focus of the experiment is to gain information on dendritic and axonal projections in brain regions. In addition, since the sections are kept in solution, a single aliquot can easily accommodate 30 to 40 sections, handling of which is less laborious, particularly in large-scale biomedical studies. Here, we illustrate how to apply the free-floating method to fluorescent immunohistochemistry staining, with a major focus on brain sections. We will also discuss how the free-floating technique can easily be modified to fit the individual needs of researchers and adapted to other tissues as well as other histochemical-based stainings, such as hematoxylin and eosin and cresyl violet, as long as tissue samples are properly fixed, typically with paraformaldehyde or formalin.

The free-floating technique allows researchers to perform histological-based stainings including immunohistochemistry on fixed tissue sections to visualize biological structures, cell type, and protein expression and localization. This is an efficient and reliable histochemical technique that can be useful for investigating a multitude of tissues, such as brain, heart, and liver.

Histologische Färbungen mit frei schwebenden Gewebeschnitten

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative ...

Histochemical Imaging of Cortical Modules in Flattened Brain Sections

Transkript

Histochemical Imaging of Cortical Modules in Flattened Brain Sections