processing of a mouse brain for downstream analysis

Processing of a Mouse Brain for Downstream Analysis

After removing brains from euthanized adult CD-1 wild-type mice flash freeze the tissue for 60 seconds in either liquid nitrogen or isopentane prechilled with dry ice, and store it at -80 degrees Celsius.
24 hours before dissecting the tissue, place a clean brain matrix on a stack of thawed freezer packs, and sandwich the sides of the matrix between two freezer packs, making sure to leave approximately half a centimeter between the bottom of the razor slots and the top of the packs.
Set up a frozen glass plate for the dissection by filling an insulated box with ice up to approximately 5 centimeters from the top. Then, place a 2.5-centimeters layer of dry ice on top and cover it with black plastic sheeting. Place a glass plate on top of the plastic and dry ice around the borders of the plate.
Remove the frozen brain matrix from the freezer and insert the brain cortex side up into the matrix. Allow the tissue to equilibrate to the temperature in the box for 10 minutes, keeping the lid open during this time. Use cold forceps to adjust the brain&#39;s position in the matrix, so that the sagittal sinus and transverse sinus line up with the perpendicular grooves of the block.
Once the brain is in position, place a chilled razor blade near its center and press it approximately 1 millimeter into the tissue. Next, place one chilled blade at each end of the brain and press all the way down into the matrix. Then, begin adding chilled blades at the rostral end, placing them into the slots one at a time and gently pressing them 1 millimeter into the tissue. Continue to add blades at 1-millimeter intervals, working toward the caudal end.
When all blades are in place, press down on top with fingers, palm, or a blunt object, and rock them slowly from side to side to move them through the tissue. Once the blades have reached the bottom of the slots, grasp each side of the group of blades, and free them from the matrix by rocking back and forth.
After freeing the group of blades, place them rostral side up on the glass plate, and put dry ice next to or on top of the stack to further freeze the samples for easier separation. Then, place the stack with sharp edges down and separate the blades by shifting the stack between the thumb and fingers.
Line up the section on the glass plates from rostral to caudal, and separate the tissue from the blades by flexing them between the fingers or by separating them with a second chilled blade. At times, tissue may stick to both sides of a blade and care must be taken to maintain rostral-to-caudal orientation.
Before starting the dissection, open the Allen Mouse Brain Atlas or another reference and find the landmarks necessary to identify regions of interest. Use chilled forceps or blades to flip the section, and make sure that the region of interest is consistent throughout the section.
Cut into the section with a clean scalpel or punch, pushing the metal gently but firmly into the tissue, rocking it back and forth to make the cut. After harvesting the region of interest, place it into labeled prechilled 1.5-milliliter tubes, and store them at -80 degrees Celsius.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Storing brains
<ol>
	<li>Quickly remove the brains from euthanized adult CD1 wildtype mice weighing approximately 30 g using a conventional approach and flash-freeze for 60 seconds in either liquid nitrogen or isopentane pre-chilled with dry ice.</li>
	<li>Store frozen brains at -80 &#176;C in aluminum foil or conical tubes until use.</li>
</ol>
2. Preparing the brain matrix
<ol>
	<li>Twenty-four hours before dissecting tissue, place a clean brain matrix on a stack of thawed freezer packs. Sandwich the sides of the matrix between two freezer packs making sure to leave approximately 0.5 cm between the bottom of the razor slots and the top of the gel packs (Figure 1A). Place aluminum foil on the ends to aid in cooling. 
	NOTE: When purchasing a brain matrix, buy one that is large enough to encase the entire volume of the brain to be dissected.</li>
	<li>Place the box containing the brain matrix and freezer packs in a -20 &#176;C freezer with the top ajar overnight.</li>
</ol>
3. Setting up a frozen glass plate
NOTE: The purpose of this setup is to prepare a frozen surface on which to dissect brain sections.
<ol>
	<li>Place ice in an insulated box up to approximately 5 cm from the top. Then place a 2.5 cm layer of dry ice on top of the ice and cover with black plastic sheeting to aid in visualizing the sample (Figure 1B, Figure 1C).</li>
	<li>Place a glass plate (should just fit in the opening of the box) on top of the plastic and place dry ice on top of the plate in the far corners (Figure 1C).</li>
	<li>Take the frozen brain matrix from the freezer and insert the brain to be cut cortex-side up. Allow it to equilibrate to the temperature in the box for 10 min. Keep the lid on the box during this time.</li>
	<li>Adjust the brain&#39;s position in the matrix with cold forceps so that the sagittal sinus and transverse sinus line up with the perpendicular grooves of the block (Figure 1D). This will help ensure symmetric sections for easier dissection. Touch tips of forceps to dry ice briefly to chill before adjusting the brain.</li>
	<li>Once the brain is in position, place a chilled razor blade near its center and press the blade approximately 1 mm into the tissue. Add chilled blades to the rostral and caudal ends to help hold the brain in place (Figure 2A and Figure 2B).</li>
	<li>Starting on the rostral end, add blades one at a time, placing them into the slots and pressing them gently down into the tissue approximately 1 mm (Figure 2B). Continue to add blades at 1 mm intervals working towards the caudal end.</li>
	<li>Make sure the brain does not shift during this time. Line up blades horizontally and vertically (Figure 2B). Low-profile microtome blades may be used to cut sections into 0.5 mm widths. Place these between the larger razor blades (Figure 2C).</li>
	<li>Once blades are in place, press down on top of the group with fingers, palm or some other flat surface (Figure 2C, Figure 2D). Rock the group of blades slowly from side to side to move them through the tissue. 
	NOTE: This may take some time (1&#8211;2 min) and requires patience. There should be resistance to the blades moving through the tissue. Easy entry means the brain is thawing and the block should be cooled with dry ice.</li>
	<li>When all blades have reached the bottom of the slots, grasp each side of the group of blades and rock back and forth to work free of the matrix. 
	NOTE: Exercise caution to avoid cutting oneself on the sharp edges of the blades.</li>
	<li>Once the group is free, place the stack, rostral side up, on the glass plate (Figure 2E). Place dry ice next to and/or on top of the stack to further freeze the samples for easier separation.</li>
	<li>Place the stack with sharp edges down on the glass plate and separate blades by shifting the stack between thumbs and fingers. The blades should separate from each other with sections attached.</li>
	<li>Line up sections on the glass plate from rostral to caudal (Figure 2F).</li>
	<li>Separate tissue from the blades by flexing blades between fingers, or by separating with a second cold razor (Figures 2G, 2H, 2I).</li>
</ol>
4. Dissecting sections
<ol>
	<li>Open the Allen Mouse Brain Atlas or another reference and find landmarks necessary to identify regions of interest. Some obvious landmarks include the anterior commissure, the corpus callosum, the lateral ventricles, and the hippocampus (Figure 3).</li>
	<li>Flip the section to be cut with chilled forceps and ensure the region about to be collected is consistent throughout. During harvesting, consult the reference atlas often to ensure the correct ROI is obtained. 
	NOTE: A magnifying glass or dissecting microscope is often helpful in this process. Good lighting is also essential. Low wattage LED lamps or a cool lamp can be used for this purpose.</li>
	<li>With a clean scalpel or punch, cut into the section (Figure 4). Prechill each tool before cutting by touching it briefly to dry ice. Push the blade gently but firmly into the tissue, rocking it back and forth to make the cut. Do not push too hard or the tissue will fracture. 
	NOTE: Tools will warm over time. Slightly warmed blades can help make clean cuts and limit fracturing, but be careful to avoid thawing tissue. Periodically chill tools on dry ice.</li>
	<li>Once an ROI is harvested, place it into labeled, prechilled 1.5 mL tubes. Store harvested tissues at -80 &#176;C until needed.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 mm Mouse coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 505C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>0.5 mm Rat coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 705C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>1.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-01</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Dot Scientific</td>
			<td>229443</td>
			<td>For storing frozen ROIs</td>
		</tr>
		<tr>
			<td>1.5 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-02</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-03</td>
			<td></td>
		</tr>
		<tr>
			<td>4-12% NuPage gel</td>
			<td>Invitrogen</td>
			<td>NPO323BOX</td>
			<td>protein gradient gel</td>
		</tr>
		<tr>
			<td>Bioanalyzer System</td>
			<td>Agilent</td>
			<td>2100</td>
			<td>RNA analysis system</td>
		</tr>
		<tr>
			<td>Dounce tissue grinder</td>
			<td>Millipore Sigma</td>
			<td>D8938</td>
			<td>Glass tissue homogenizer</td>
		</tr>
		<tr>
			<td>Fiber-Lite</td>
			<td>Dolan-Jenner Industries Inc.</td>
			<td>Model 180</td>
			<td>Cool lamp</td>
		</tr>
		<tr>
			<td>Glass plates</td>
			<td>LabRepCo</td>
			<td>11074010</td>
			<td></td>
		</tr>
		<tr>
			<td>Low profile blades</td>
			<td>Sakura Finetek USA Inc.</td>
			<td>4689</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Wager-Miller, J. et al.&#160; <a href="https://app.jove.com/v/60474">Collection of Frozen Rodent Brain Regions for Downstream Analyses.</a> J. Vis. Exp (2020)
This video demonstrates a detailed step-by-step protocol for preparing frozen mouse brain tissue sections for downstream analyses.

<img alt="Figure 1" src="/files/ftp_upload/23217/23217_Figure1.jpg" /> 
Figure 1: Preparing frozen work surfaces. (A) A brain block is placed on a stack of gel packs within an insulated box and is then sandwiched between two additional gel packs. Aluminum foil is packed at each end of the block to facilitate cooling during sectioning. The box is then cooled overnight at -20 &#176;C. (B) A glass plate is matched for size with the open...

Source: Wager-Miller, J. et al.&#160; Collection of Frozen Rodent Brain Regions for Downstream Analyses. J. Vis. Exp (2020)
This video demonstrates a ...

Take a freshly harvested mouse brain and flash-freeze it in pre-chilled isopentane for rapid tissue preservation.&#160;&#160; &#160;&#160;
Next, transfer the brain, cortex side up, into a frozen brain matrix to maintain a cold environment during slicing.
Allow the brain to equilibrate to the matrix temperature. Align it in the matrix along the sagittal and transverse sinuses to ensure symmetrical sections.
Insert chilled blades into the brain, ensuring proper alignment.
Then, press them down evenly to obtain uniform brain sections.
Remove the blades with the sections and position them, rostral side up, on a frozen glass plate.
Organize the sections from rostral to caudal orientation in the correct anatomical order. Now, separate them.
Using a suitable brain reference, locate and excise the region of interest from the section.
Transfer the excised tissue into a pre-chilled vial for downstream analysis.

12 visualizações. Processing of a Mouse Brain for Downstream Analysis

Vídeo: Processing of a Mouse Brain for Downstream Analysis - Experimento

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Aplicação de correia de patch automatizada com guia de imagem para estudo de neurônios em fatias de cérebro

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

JoVE Journal

Neurociência

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Combinando Hibridização In Situ por Fluorescência Multiplex com Imunohistoquímica Fluorescente em Seções Cerebrais de Camundongos Frescos Congelados ou Fixos

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

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.

Coloração histológica usando seções de tecido flutuantes livres

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

Processing of a Mouse Brain for Downstream Analysis

Transcrição

Processing of a Mouse Brain for Downstream Analysis