immunostaining of interneurons in a rat hippocampal section

Immunostaining of Interneurons in a Rat Hippocampal Section

Lock the tissues with 10% blocking serum for one hour at room temperature. Subsequently, incubate the sections in primary antibody CB1R at room temperature overnight, to identify cannabinoid receptor type one expression. Then, wash the brain sections three times for 10 minutes each in 0.1 molar PBS. Streptavidin and secondary antibody to first primary antibody, are both added to the well plate and kept overnight at four degrees Celsius.
Subsequently, incubate the sections in streptavidin red dye conjugate at four degrees Celsius overnight in the dark to reveal the biocytin. After that, wash the brain sections three times for 10 minutes each in 0.1 molar PBS. To protect the fluorescence and to facilitate restaining in the future, mount the sections in an aqueous-based mounting medium, and seal the edges of the coverslips with clear nail polish.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;
1. Staining of the Primary Antibody and Biocytin
(Day 1) 
NOTE: The following immunostaining procedure is for free-floating sections and requires continuous shaking at a low speed (2 revolutions/min, rpm) on a shaker for all incubation steps.
<ol>
	<li>Wash the tissue 3x for 10 min each in 0.1 M PBS (pH = 7.4). 
	NOTE: 0.1 M PBS can be commercially purchased or made in the laboratory as follows (makes 1 L): 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 1 L of dH2O. 
	NOTE: If the desired neurochemical marker is known, staining for a protein/peptide can be performed simultaneously with biocytin staining (steps 1.2 - 1.4). If staining for biocytin only, proceed to step 1.5.</li>
	<li>OPTIONAL: Block with 10% Blocking Serum (BS; normal goat serum (NGS) diluted in 0.3% Triton X-100 in PBS for 1 h at RT). 
	NOTE: Each well should receive the same volume of the solution; the recommended volume is between 250 - 500 &#181;L/well. The selection of the blocking serum is based on the species in which the secondary antibodies are raised (e.g., NGS is used for dye-conjugated goat anti-(respective primary antibody species)). If the need arises to use secondary antibodies raised in different species to immunostain sections, include 10% of each normal serum in blocking step (2) and 3% of each normal serum for the primary and secondary antibody incubation steps.</li>
	<li>(OPTIONAL) Incubate the sections in primary antibody, CB1R (polyclonal, guinea pig), with 0.3% Triton X-100 and 3% BS in 0.1 M PBS at RT O/N. CB1R is used to identify cannabinoid receptor type 1 expression. 
	NOTE: This step is optional and not required to reveal the biocytin filling. Figure 2 illustrates a section labeled for biocytin and CB1R during the first staining process. O/N incubation at RT is sufficient for most of the primary antibodies; however, certain primary antibodies, including CB1R, require 3 - 5 d of incubation. If the incubation needs to be longer than 1 d, it is recommended to incubate at 4 &#176;C because Triton X-100 can permeabilize slices and can lead to the disintegration of the tissue during prolonged incubation at room temperature. Include a negative control lacking a primary antibody in at least one section for each series of experiments as a control for the specificity of the secondary antibody. For negative controls, the primary antibody is omitted, but the 0.3% Triton X-100 in 0.1 M PBS and BS are added.</li>
</ol>
(Day 2)
<ol start="4">
	<li>Wash the brain sections 3x for 10 min each in 0.1 M PBS (pH = 7.4).</li>
	<li>Incubate the sections in streptavidin red dye conjugate (concentration: 1:1,000; excitation: 594 nm with emission in the visible red spectrum) with 0.3% Triton X-100 and 3% BS in 0.1 M PBS at 4 &#176;C O/N in the dark (wrapped in aluminum foil) to reveal the biocytin. OPTIONAL: Include a secondary antibody, goat anti-guinea pig (concentration: 1:500; excitation: 488 nm and emission in green), with the above incubation following the optional primary incubation in step 1.3. 
	NOTE: A secondary antibody will be needed only if the optional primary antibody staining (step 1.3) is performed. To visualize biocytin, a streptavidin-fluorophore conjugate with an emission spectrum in red is recommended, as it helps visualize finer axons in detail and with better contrast (Figures 1 - 3).</li>
</ol>
(Day 3)
<ol start="6">
	<li>Wash brain sections 3x for 10 min each in 0.1 M PBS (pH = 7.4).</li>
	<li>To protect the fluorescence and to facilitate re-staining in the future, mount the sections in an aqueous-based mounting medium and seal the edges of the coverslip with clear nail polish. 
	NOTE: It is best to restain sections as early as possible to avoid difficulties removing the nail polish from the slide, mold infestation, and tissue dehydration due to the mounting medium leaking from the glass slide. Microbial contamination can be prevented by using 0.02% sodium azide in PBS. 
	CAUTION: Sodium azide is highly toxic and flammable when it is in contact with water. Please refer to standard operating procedures for the handling and disposal of hazardous chemicals. 
	(Days 4 - 7)</li>
	<li>Perform confocal imaging with excitation at 594 and 488 nm and at a magnification of 20X or 40X to reveal biocytin-filled neurons in red and neurochemical markers (CB1R) in green. Assess colabeling (Figure 2).</li>
	<li>Set the camera exposure to less than 100 ms to avoid photobleaching and obtain confocal image stacks of the axonal and dendritic arbors of the entire neuron. Use confocal image stacks and neuron tracing software packages to reconstruct the neuronal morphology.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>Immunostaining</td>
		</tr>
		<tr>
			<td>KCL</td>
			<td>Fluka</td>
			<td>60129</td>
			<td>Immunostaining</td>
		</tr>
		<tr>
			<td>Disodium hydrogen phosphate</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td>Immunostaining</td>
		</tr>
		<tr>
			<td>Monopotassium phosphate</td>
			<td>Sigma</td>
			<td>229806</td>
			<td>Immunostaining</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Immunostaining</td>
		</tr>
		<tr>
			<td>Guinea pig anti CB1</td>
			<td>Sigma</td>
			<td>Af530-1</td>
			<td>Immunostaining</td>
		</tr>
		<tr>
			<td>Streptavidin, Alexa Fluor conjugate</td>
			<td>Molecular Probes</td>
			<td>S11227</td>
			<td>Immunostaining</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td>Immunostaining</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td>Immunostaining</td>
		</tr>
		<tr>
			<td>Secondary Antibodies</td>
			<td>Invitogen Molecular probes</td>
			<td>Alexa Fluor conjugated dyes</td>
			<td>Immunostaining</td>
		</tr>
		<tr>
			<td>Labnet orbit low speed shaker</td>
			<td>Bioexpress</td>
			<td>S-2030-LS</td>
			<td>Immunostaining</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td>11231-30</td>
			<td>Immunostaining</td>
		</tr>
		<tr>
			<td>Slide folders</td>
			<td>EMS</td>
			<td>71520</td>
			<td>Immunostaining</td>
		</tr>
		<tr>
			<td>Biocytin</td>
			<td>Sigma</td>
			<td>B4261</td>
			<td>Electrophysiology</td>
		</tr>
	</tbody>
</table>

Swietek, B., et al., <a href="https://app.jove.com/v/54880">Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers.</a> J. Vis. Exp. (2016)
This video demonstrates the process of visualizing a biocytin-filled interneuron in a rat hippocampal section, highlighting its neurochemical identity through the use of specific antibodies and fluorescence microscopy.

<img alt="Figure 1" src="/files/ftp_upload/23111/23111_Figure1.jpg" /> 
Figure 1. Suggested Plane for Approaching Cells for Biocytin Fills. The image illustrates a Granule Cell (GC) filled with biocytin during recording that has been processed to reveal biocytin (in red using 594-conjugated streptavidin). The GC has its dendrites oriented in the XY plane. Notice the severed granule cell axon (green arrow) due to pipette movement along the XY plane. Note that it is id...

Swietek, B., et al., Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers. J. Vis. Exp. (2016)
This video demonstrates the ...

Take a fixed rat hippocampal section containing an interneuron filled with biocytin, a biotin-conjugated molecule.&#160;
Next, add a solution containing blocking proteins and a non-ionic detergent to prevent non-specific binding and permeabilize neurons.
Incubate with a primary antibody targeting specific cannabinoid receptors on interneurons.
Wash with buffer to remove excess antibodies.
Then, add a streptavidin-conjugated fluorescent dye and a fluorophore-conjugated secondary antibody.
During incubation, streptavidin binds strongly to biocytin, allowing visualization of the biocytin-filled interneuron, while the secondary antibody targets receptor-bound primary antibodies on the interneurons.
Rinse with buffer, removing unbound molecules.
Now, mount the section using aqueous mounting media. Position a coverslip and seal it.
Using a fluorescence microscope, visualize the biocytin-filled interneuron and cannabinoid receptor expression, enabling analysis of the interneuron&#39;s morphology and receptor distribution.

5 visualizações. Immunostaining of Interneurons in a Rat Hippocampal Section

Vídeo: Immunostaining of Interneurons in a Rat Hippocampal Section - Experimento

Biocytin Recovery and 3D Reconstructions of Filled Hippocampal CA2 Interneurons

How cortical network activity processes information is of importance to a large number of basic and clinical scientific questions. The protocol described here identifies the basic building blocks of this circuitry. The in-depth studies of cortical regions will ultimately provide other scientists with the circuit components needed for an understanding of how the brain acquires, processes and stores information and what goes wrong in disease, while the electrophysiological and morphological data are widely used by computational neuroscientists in the construction of model networks that explore information processing. The protocol outlined here describes how biocytin-filled cells recorded in the CA2 region of the hippocampus are recovered and then reconstructed in&#160;3D. Additionally, the protocol describes the demonstration of calcium binding protein or peptide content in recorded interneurons.

The protocol outlined here describes the immunofluorescence analysis, biocytin recovery and high-quality reconstructions of hippocampal CA2 interneurons following the intracellular electrophysiological recordings in vitro, allowing neuronal characterization and ultimately fine neuronal anatomy to be studied.

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Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

Immunostaining of Interneurons in a Rat Hippocampal Section

Transcrição

Immunostaining of Interneurons in a Rat Hippocampal Section