labeling of biocytin-filled interneurons in rat hippocampal slices for visualization

Labeling of Biocytin-Filled Interneurons in Rat Hippocampal Slices for Visualization

Place the sections into a clean glass vial containing PBS. Perform the avidin HRP reaction by first incubating the sections in avidin biotin complex, or ABC, for at least two hours to amplify the HRP reaction product.
Next, wash the sections with PBS 3 times for 10 minutes, and then with Tris buffer twice for 10 minutes. After removing the last trace buffer wash, quickly add 1 drop of 8% nickel chloride solution to the diaminobenzidine solution, or DAB solution. Then pipette the solution in and out to mix.
And quickly add 1 milliliter of this solution over the sections. Incubate the sections in this solution for 15 minutes. Then, add 10 microliters of 1% hydrogen peroxide to the DAB solution. Allow the reaction to proceed in the dark under constant agitation for about 1 to 2 minutes, until the cells are labeled.
In a fume hood, place a small circle of filter paper into a Petri dish and dampen it with PB. Lift the sections one at a time from the glass vial using a paintbrush, and carefully place them flat on the paper. Cover the sections with another moistened circle of filter paper. And remove excess buffer by gently touching tissue paper to the surface.
Apply 8 or 9 drops of 1% osmium tetroxide in PB to the top paper. Cover the dish and retain in the fume hood for at least 30 minutes, but no more than one hour. After rinsing, mounting, and cover-slipping the sections, dehydrate through a series of 50%, 70%, 95%, and 100% alcohol solutions.
Following the dehydration step, transfer the sections to a glass vial containing 100% ethanol on a shaker for approximately 5 minutes. Replace the alcohol solution with propylene oxide, and wash three times for 5 minutes. Following the last wash, keep around 2 milliliters of propylene oxide in the vial, and add resin at a one-to-one ratio.
Ensure that the resin is dissolved, and keep the sections under constant agitation for 30 minutes. After 30 minutes, use a wooden stick to place each section in an aluminum planchet containing epoxy resin and incubate overnight. The next day, place the planchet on a hot plate for approximately 10 minutes.
Then, pick up each section with a wooden stick and place on a clean slide. Once all the sections have been transferred onto the slide, view the slide under a dissecting microscope to check the orientation of the slices and place a coverslip over the sections. Cure in the oven for 48 hours at 56 degrees Celsius.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee
1. Determination of Calcium Binding Protein or Protein Content of Interneurons and Biocytin Visualization Following Electrophysiological Recordings and Biocytin Filling
Note: At the end of the electrophysiological recordings, slices that contain biocytin-filled cells are fixed overnight prior to histological procedures. The fixative solution will be replaced with a single change of 0.1 M phosphate buffer the next morning if the rest of the procedure is carried out on another day in order to prevent tissue damage. The fixation solution (4% paraformaldehyde, 0.2% saturated picric acid solution, 0.025% glutaraldehyde solution in 0.1 M phosphate buffer (PB)) must be made fresh on the day of the recordings for best results.
<ol>
	<li>At the end of the electrophysiological recording, carefully pick up the slice containing the recorded cell(s) with a paintbrush and place it in a pot containing artificial cerebral spinal fluid (ACSF). 
	NOTE: Details of the protocol used for slice preparation and intracellular recordings using sharp electrodes have been described previously.</li>
	<li>In a fume hood, carefully pick up the brain slice with a paintbrush and roll it onto a small piece of fine-quality filter paper. Place another piece of moistened filter paper onto the slice and place the two pieces of wet filter paper in a small plastic pot containing 5-10 mL of fixative solution and store in the fridge overnight at 4 &#176;C.</li>
	<li>Prepare a solution of gelatin in distilled water. Place 20 mL of distilled water in a beaker on a hot plate and heat it to 60 &#176;C. Add progressively 2.4 g of gelatin to the water, wait until dissolved, and allow it to cool to 35&#8211;40 &#176;C before use to prevent tissue damage.</li>
	<li>Replace the fixative solution with 2 mL of 0.1 M PB. Place the slice in a Petri dish and cut away any excess tissue with a scalpel blade. Place the trimmed tissue in a Petri dish (diameter of 9 cm, height of 1.4 cm), ensuring that it lies flat with no folds or creases, and remove the excess buffer using a dry paintbrush.</li>
	<li>Cover the tissue with the warm gelatin solution and place the Petri dish onto a frozen block to quickly cool the solution.</li>
	<li>Using an angle-poise lamp to provide contrast, look through the side of the dish and keep the slice flat using light pressure from a fine paintbrush until the gelatin begins to set. 
	NOTE: The gelatin can be re-melted if the tissue does not lie flat. Any imperfections on the surface of the gelatin caused by the removal of the paintbrush as it solidifies may be removed by melting the surface layer gently by moving the bulb of the lamp close to the surface for a few seconds.</li>
	<li>Move the dish of setting gelatin to the fridge and leave at 4 &#176;C for 30-60 min.</li>
	<li>In a fume hood, cut out a small block (~ 1 x 1 cm) containing the gelatin-embedded tissue of the dish using a scalpel blade, lift the block using a small spatula, and carefully place it in the same but fresh fixative solution used to fix the slices for at least 30 min at 4 &#176;C.</li>
	<li>Wash the gelatin block in 5 mL of 0.1 M PB three times, dry it using a piece of paper tissue, and stick the block side up (i.e., with the tissue at the top) onto a vibratome chuck using super glue.</li>
	<li>Remove excess glue with a piece of filter paper and use a scalpel blade to cut the corners of the block off, leaving a diamond shape.</li>
	<li>Section the slice at 50 &#181;m thickness using a vibratome and place each section carefully in a glass vial containing 10% sucrose.</li>
	<li>Carefully pick up a section from the vial, and place it flat into a Petri dish lid. Using a dissecting microscope and a fresh scalpel blade, remove the gelatin from around the section and return the section to a vial containing 2 mL of fresh 10% sucrose 
	NOTE: It is crucial to remove as much gelatin as possible at this stage to reduce tissue shrinkage during the dehydration step (Step 1.37).</li>
	<li>Cryo-protect the sections in 0.1 M PB-based sucrose-glycerol solution at room temperature by incubating them for 10 min in 10% sucrose solution, 20 min in 20% sucrose-6% glycerol solution twice, and finally 30 min in 30% sucrose-12% glycerol solution twice under constant agitation.</li>
	<li>Place the sections flat onto a small rectangle of tin foil using a paintbrush. Remove any excess liquid from the sections and carefully fold the tin foil into a parcel.</li>
	<li>Hold the parcel close to the surface of liquid nitrogen without touching the surface for 30 s and then allow the sections to thaw completely for approximately 30 s. Repeat the freeze-thaw another two times.</li>
	<li>Remove all sections with a paintbrush and place them in a glass vial containing 2 mL of 0.1 M PB under constant agitation to wash off excess sucrose.</li>
	<li>Remove the PB with a Pasteur pipette and incubate the sections in 2 mL of 1% aqueous hydrogen peroxide (H2O2) for 30 min. Wash the sections in 2 mL of 0.1 M PB 3x 5 min.</li>
	<li>Remove the 0.1 M PB with a Pasteur pipette and add 1 % sodium borohydride (NaBH4) in 0.1 M PB. 
	NOTE: Do not cap the vial, as NaBH4 solution gives off hydrogen gas.</li>
	<li>Remove the sodium borohydride with a Pasteur pipette and wash the sections thoroughly in 2 mL of 0.1 M PB 5x 5 min.</li>
	<li>Replace the 0.1 M PB with 10% normal goat serum (NGS) in 0.1 M PB for 30 min.</li>
	<li>Remove the goat serum and incubate the sections overnight at 4 &#176;C in a mixture of mouse monoclonal and rabbit polyclonal antibodies made up in ABC solution. 
	NOTE: A list of primary antibodies used in previous studies is displayed in Table 1.</li>
	<li>Incubate the sections for 2 h in the dark in a mixture of fluorescently labeled secondary antibodies (Table of Materials).</li>
	<li>Mount the sections onto the slides in mounting medium and cover them with a coverslip.</li>
	<li>Take images of fluorescence labeling at 40X magnification (Figure 1Bb).</li>
	<li>After the fluorescence imaging, place the slide into a glass Petri dish containing PBS and carefully remove the coverslip. Then wash the sections off the slide using gentle pulses of PBS from a Pasteur pipette. Place the sections into a clean glass vial containing PBS.</li>
	<li>Perform the avidin-HRP reaction by firstly incubating the sections in ABC for at least 2 h to amplify the HRP reaction product.</li>
	<li>Prepare the 3,5 diaminobenzidine (DAB) solution by adding one tablet to 5 mL of distilled water.</li>
	<li>Wash the sections with PBS three times for 10 min and then with Tris buffer twice for 10 min. Remove the Tris buffer after the last wash.</li>
	<li>Quickly add one drop of 8 % NiCl2 (Nickel chloride) solution to the DAB solution, pipette the solution in and out to mix, and quickly add 1 mL of this solution over the sections. Incubate the sections in the DAB/NiCl2 solution for 15 min.</li>
	<li>Add 10 &#181;L of 1% H2O2 to the DAB solution. Allow the reaction to proceed in the dark under constant agitation for about 1 to 2 min, and monitor the labeling of the filled cells with a dissecting microscope.</li>
	<li>Stop the reaction by removing the DAB/NiCl2/H2O2 solution and wash the sections with Tris buffer twice for 5 min.</li>
	<li>In a fume hood, place a small circle of filter paper into a Petri dish and dampen it with 0.1 M PB. Lift the sections one at a time from the glass vial using a paintbrush, and place them carefully flat upon the paper.</li>
	<li>Cover the sections with another moistened circle of filter paper and remove excess buffer by gently touching tissue paper to the surface.</li>
	<li>Apply 8&#8211;9 drops of 1% osmium tetroxide in 0.1 M PB to the top paper, cover the dish, and retain in the fume hood for at least 30 min, but no more than 1 h.</li>
	<li>Open the Petri dish and lift the top filter paper. Lift the sections carefully one at a time with a paintbrush, place them in a glass vial, and rinse them in distilled water twice.</li>
	<li>Dispose of osmium tetroxide waste appropriately. Rinse all disposable equipment and place them in appropriate bins.</li>
	<li>Place each section flat onto a glass slide and coverslip the sections. Transfer the slide into a Petri dish, place an empty glass vial over the coverslip to retain it in place, and cover with 50% alcohol. After 15 min, remove the slide from the solution and remove the sections from the slide. Place the sections back on the slide and then place the slide in 70% alcohol for 15 min. Repeat the same process with 95% and finally 100% alcohol solution.</li>
	<li>Following the dehydration step, transfer the sections to a glass vial containing 100% alcohol on a shaker in a fume hood. Replace the alcohol solution with propylene oxide (C3H6O) and wash three times for 5 min. Following the last wash, keep ~2 mL of propylene oxide in the vial and add resin (1:1 ratio). Ensure that the resin is dissolved and keep the sections under constant agitation for 30 min.</li>
	<li>Place each section in an aluminum planchette containing epoxy resin using a wooden stick and incubate overnight. 
	NOTE: Do not leave the sections in the resin longer than 24 h to avoid the risk of damaging the sections.</li>
	<li>Place the planchette over a hot plate for approximately 10 min. Pick up each section with a wooden stick and place them on a clean slide. Keep the orientation of each section consistent using a dissecting microscope. Place a coverslip over the sections. Place the slide in the oven for 48 h at 56 &#176;C for curing.</li>
</ol>
Table 1: Table of solutions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solutions used </td>
			<td>Composition/Instructions</td>
		</tr>
		<tr>
			<td>Fixation solution</td>
			<td>4% paraformaldehyde, 0.2% saturated picric acid solution, 0.025% glutaraldehyde solution in 0.1 M phosphate buffer (PB)</td>
		</tr>
		<tr>
			<td>0.1M Phosphate Buffer pH 7.6</td>
			<td>Add 100 mL of stock 1 M Phosphate Buffer to 900 mL of distilled water</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) pH 7.4/7.5</td>
			<td>Add 10 mL of 0.1M phosphate buffer, 0.2 g of KCl, and 8.76 g of NaCl to 990 mL of distilled water</td>
		</tr>
		<tr>
			<td>TRIS buffer pH 7.5</td>
			<td>Dissolve 5.72 g of Tris Hydrochloride and 1.66 g of Tris Base in 50 mL of distilled water. Then make up to 1 L with distilled water.</td>
		</tr>
		<tr>
			<td>Buffered glutaraldehyde and paraformaldehyde fixative solution</td>
			<td>4% paraformaldehyde, 0.2% saturated picric acid solution, 0.025% glutaraldehyde solution in 0.1 M Phosphate buffer.</td>
		</tr>
		<tr>
			<td>ABC solution</td>
			<td>Solution to be made at least 30 min before use from the ABC kit. Add 1 drop of solution A and 1 drop of solution B to 2.5 mL of PBS.</td>
		</tr>
		<tr>
			<td>Durcupan epoxy resin:</td>
			<td>To make 20 pots: 20 g of component A, 20 g of component B, 0.6 g of component C and 0.4 g of component D-</td>
		</tr>
		<tr>
			<td></td>
			<td>Protect the balance from spills by covering the plate with a circle of filter paper. Carefully weigh the reagents into a tripour beaker in the proportions stated above. Mix thoroughly by vigorously stirring using two wooden sticks for at least 5 min. The mixture should become a uniform density dark brown color. Place the beaker into the oven at ~50 &#176;C for a maximum of 10 min to remove as many air bubbles as possible. NOTE: The resin will start to cure if you leave the beaker in the oven longer than 10 min. Decant the resin out into plastic pots or 5 mL syringes, date them, and store them in the -20 &#176;C freezer ready for use.</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Avidin-7-amino-4-methylcoumarin-3-acetic acid (Avidin-AMCA) antibody- 20.8 mg/mL</td>
			<td>Vector laboratories</td>
			<td>A2008</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocytin &#8805;98% (TLC)</td>
			<td>Sigma</td>
			<td>B4261</td>
			<td></td>
		</tr>
		<tr>
			<td>3,5 diaminobenzidine (DAB) tablet, To prepare 5 mL</td>
			<td>Sigma</td>
			<td>D4293</td>
			<td></td>
		</tr>
		<tr>
			<td>Durcupan epoxy resin A</td>
			<td>Sigma</td>
			<td>44611</td>
			<td></td>
		</tr>
		<tr>
			<td>Durcupan epoxy resin B</td>
			<td>Sigma</td>
			<td>44612</td>
			<td></td>
		</tr>
		<tr>
			<td>Durcupan epoxy resin C</td>
			<td>Sigma</td>
			<td>44613</td>
			<td></td>
		</tr>
		<tr>
			<td>Durcupan epoxy resin D</td>
			<td>Sigma</td>
			<td>44614</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, puriss. p.a., absolute, &#8805;99.8%</td>
			<td>Sigma</td>
			<td>32221</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>48723</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde solution, grade I, 25 % in H2O</td>
			<td>Sigma</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol, &#8805;99%</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse fluorescein isothiocyanate (FITC)- 14.3 mg/mL</td>
			<td>Sigma</td>
			<td>F2653</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Texas Red (TR)- 3.3 mg/mL</td>
			<td>Invitrogen</td>
			<td>T2767</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide, 30% solution</td>
			<td>Sigma</td>
			<td>H-1009</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil, viscosity 1.250 cSt (lit.)</td>
			<td>Sigma</td>
			<td>I0890</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel chloride</td>
			<td>Sigma</td>
			<td>N5756</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide, for electron microscopy, 4% in water</td>
			<td>Sigma</td>
			<td>75632</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, reagent grade, crystalline</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Picric acid, moistened with water, &#8805;98%</td>
			<td>Sigma</td>
			<td>197378</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer 1 M</td>
			<td>Sigma</td>
			<td>P3619</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene oxide, 99%</td>
			<td>Alfa Aesar</td>
			<td>30765</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tetrahydroborate</td>
			<td>VWR</td>
			<td>27885.134</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher scientific</td>
			<td>S/8600/53</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Hydrochloride, &#8805;99.0%</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base, &#8805;99.9%</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield Antifade Mounting Medium, , refractive index 1.45</td>
			<td>Vector laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain Elite ABC HRP kit</td>
			<td>Vector laboratories</td>
			<td>PK6100</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment used</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Agar Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C4A Cupped aluminium planchettes</td>
			<td>GA-MA &#38; ASSOCIATES, INC.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMR microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X-Cite 120PC Q fluorescence light source</td>
			<td>Excelitas Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DFC450 digital microscope camera</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rapidograph technical drawing pen 0.18mm</td>
			<td>London graphics centre</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.18mm rapidograph nib</td>
			<td>London graphics centre</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rapidograph technical drawing pen 0.25mm</td>
			<td>London graphics centre</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25mm rapidograph nib</td>
			<td>London graphics centre</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurolucida system including PC workstation, stage, camera and joystic for XYZ stage control</td>
			<td>Microbrighfield (MBF) Bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurolucida software version 2017</td>
			<td>Microbrighfield (MBF) Bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CorelDRAW graphics Suite X5</td>
			<td>Corel</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Video editing software</td>
			<td>Adobe Premiere Pro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vials 14 mL</td>
			<td>Fisher scientific</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Economides, G., et al. <a href="https://app.jove.com/v/58592">Biocytin Recovery and 3D Reconstructions of Filled Hippocampal CA2 Interneurons.</a> J. Vis. Exp. (2018)
This video demonstrates the procedure for staining fixed rat brain slices containing biocytin-filled interneurons. The protocol includes incubation with avidin-biotin complex (ABC), staining with HRP and chromogenic substrate, preserving structures with osmium tetroxide, and embedding the tissue in resin for imaging.

<img alt="Figure 1" src="/files/ftp_upload/23197/23197_Figure1.jpg" /> 
Figure 1: Neuronal reconstructions of two types of basket cells recorded and filled in the hippocampal CA2 region and correlated electrophysiological data obtained following intracellular recordings in vitro. This figure has been modified from previous studies. SO Stratum Oriens, SP Stratum Pyramidale, SR Stratum Radiatum, SLM Stratum Lacunosum Moleculare. (A</st...

Source: Economides, G., et al. Biocytin Recovery and 3D Reconstructions of Filled Hippocampal CA2 Interneurons. J. Vis. Exp. (2018)
This video ...

Take fixed and permeabilized rat hippocampal slices containing biocytin-filled interneurons with inactivated endogenous peroxidases.
Add avidin-biotin complexes (ABC) containing horseradish peroxidase (HRP) and incubate, allowing the avidin to bind to biocytin.
Wash off unbound ABC.
Add a mixture of chromogenic substrate and metal enhancer, then incubate.
Introduce hydrogen peroxide to activate the HRP, which oxidizes the substrate to form a dark precipitate, while the enhancer improves contrast.
Transfer the slices to filter paper and remove excess buffer. Treat with osmium tetroxide to enhance the contrast.
Place the slices on a slide and mount. Dehydrate them using increasing ethanol concentrations.
Wash the slices in absolute ethanol and propylene oxide.
Add resin to infiltrate the tissues.
Place the slices into a planchette and heat to polymerize the resin. Transfer the slices to a slide, place a coverslip, and solidify the resin.
Using light microscopy, visualize the darkly stained biocytin-filled interneurons.

8 조회수. Labeling of Biocytin-Filled Interneurons in Rat Hippocampal Slices for Visualization

비디오: Labeling of Biocytin-Filled Interneurons in Rat Hippocampal Slices for Visualization - 실험

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

쥐 중추 신경계에서 면역 조직 화학 분석 및 주변 림프절 조직 섹션

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

연구

JoVE Journal

신경과학

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

신경 화학적 마커에 대한 Biocytin 충전 및 가공 섹션의 면역 염색

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

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.

자유 부유 조직 절편을 사용한 조직학적 기반 염색

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

Labeling of Biocytin-Filled Interneurons in Rat Hippocampal Slices for Visualization

내레이션 대본

Labeling of Biocytin-Filled Interneurons in Rat Hippocampal Slices for Visualization