fluorescence immunostaining of rat hippocampal neurons within a microfluidic chip

Fluorescence Immunostaining of Rat Hippocampal Neurons within a Microfluidic Chip

To begin fluorescence immunostaining, remove most of the media from the chip, keeping the interior compartments hydrated. Immediately, add 100 microliters of fixation solution to the top wells of the axonal and somatic compartments.
After one minute, add 100 microliters of fixation solution to the bottom wells, and fix the cells for 30 minutes at room temperature. Remove most of the solution from the wells, and immediately add 150 microliters of PBS to each of the top wells of the axonal and somatic compartments. Wait two minutes for the PBS to flow into the bottom wells, then, remove the PBS and rinse the wells twice more using this same process.
Following the last rinse, remove most of the PBS from the wells of the chip, and immediately add 150 microliters of PBS with 0.25% Triton X-100 to each of the top wells of the axonal and somatic compartments.
Wait for 15 minutes, and then, remove most of the liquid from the wells. Immediately add 150 microliters of blocking solution to each of the top wells of the axonal and somatic compartments. After 15 minutes, remove most of the liquid from the wells, and immediately add 100 microliters of the primary antibody solution to each of the top wells of the axonal and somatic compartments.
Cover the chip to minimize evaporation, and incubate the cells in the primary antibody for one hour at room temperature. Next, rinse the chip by removing most of the solution, and immediately adding 150 microliters of PBS to each of the top wells of the axonal and somatic compartments. After five minutes, remove the PBS from both wells, and repeat the rinse two more times.
Now, remove most of the liquid from the wells, and immediately add 100 microliters of secondary antibody in PBS to each of the top wells of the axonal and somatic compartments. Cover the chip to minimize evaporation, and incubate the chip for one hour at room temperature. As previously shown, rinse the chip three times with PBS. Then, mount and image the chips as described in the accompanying text protocol.

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1. Fluorescence Immunostaining within the Chip
<ol>
	<li>Prepare 4% formaldehyde fixation solution in PBS (4% formaldehyde, 1 &#181;M MgCl2, 0.1 &#181;m CaCl2, 120 mM sucrose)</li>
	<li>Remove most of the media in the wells of the chip (do not dry interior compartments).</li>
	<li>Immediately add 100 &#181;L of fixation solution to the top wells of the axonal and somatic compartments.</li>
	<li>After 1 min, add 100 &#181;L of fixation solution to the bottom wells. Fix for 30 min at room temperature.</li>
	<li>Remove most of the solution from the wells of the chip (do not dry interior compartments). Immediately add 150 &#181;L of PBS to each of the top wells of the axonal and somatic compartments. Wait 2 min for the PBS to flow into the bottom wells.</li>
	<li>Repeat step 1.5 twice.</li>
	<li>Remove most of the PBS from the wells of the chip. Immediately add 150 &#181;L of PBS with 0.25% TritonX-100 to each of the top wells of the axonal and somatic compartments. Wait for 15 min.</li>
	<li>Remove most of the liquid from the wells of the chip and immediately add 150 &#181;L of blocking solution (10% normal goat serum in PBS) to each of the top wells of the axonal and somatic compartments. Wait for 15 min. 
	NOTE: Effective blocking solutions should be specific to the secondary antibody, e.g., for a donkey anti-sheep secondary antibody, use donkey serum in the blocking solution.</li>
	<li>Remove most of the liquid from the wells of the chip and immediately add 100 &#181;L of primary antibody (or antibodies) in 1% normal goat serum in PBS to each of the top wells of the axonal and somatic compartments. Cover to minimize evaporation and wait for 1 h at room temperature or 4 &#176;C overnight.</li>
	<li>Remove most of the solution from the wells of the chip (do not dry interior compartments). Immediately add 150 &#181;L of PBS to each of the top wells of the axonal and somatic compartments. Wait 5 min for the PBS to flow into the bottom wells.</li>
	<li>Repeat step 1.10 twice.</li>
	<li>Remove most of the liquid from the chip&#39;s wells and immediately add 100 &#181;L of secondary antibody (or antibodies) in PBS to each of the top wells of the axonal and somatic compartments. Cover to minimize evaporation and wait for 1 h at room temperature. 
	NOTE: Refer to the manufacturer&#39;s instructions for the recommended dilution of secondary antibodies.</li>
	<li>Repeat steps 1.10-1.11.</li>
	<li>If imaging within 1 day of immunostaining, keep the chip filled with PBS. If the chip will be stored longer than 1 day before imaging, wrap the dish containing the chip in paraffin film to prevent evaporation and store it at 4 &#176;C until ready to image.</li>
	<li>For longer-term storage of samples, mounting media (e.g., Fluoromount-G) can be used.
	<ol>
		<li>Remove most of the liquid from the wells of the chip. Use a 1 mL disposable plastic pipet to add 2 drops of mounting media to each of the top wells of the axonal and somatic compartments.</li>
		<li>Tilt the chip to encourage the flow of the mounting media through the channels. After 5 min, add 2 drops to the bottom wells. Wait for 1 h before imaging. 
		NOTE: After using mounting media it will not be possible to re-probe for other targets.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>XonaChip</td>
			<td>Xona Microfluidics, LLC</td>
			<td>XC150</td>
			<td>150 &#181;m length microgroove barrier</td>
		</tr>
		<tr>
			<td>XonaChip</td>
			<td>Xona Microfluidics, LLC</td>
			<td>XC450</td>
			<td>450 &#181;m length microgroove barrier</td>
		</tr>
		<tr>
			<td>XonaChip</td>
			<td>Xona Microfluidics, LLC</td>
			<td>XC900</td>
			<td>900 &#181;m length microgroove barrier</td>
		</tr>
		<tr>
			<td>XC pre-coat</td>
			<td>Xona Microfluidics, LLC</td>
			<td>XC Pre-Coat</td>
			<td>included with XonaChips</td>
		</tr>
		<tr>
			<td>XonaPDL</td>
			<td>Xona Microfluidics, LLC</td>
			<td>XonaPDL</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>ThermoFisher Scientific</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipettes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS7095D5X SIGMA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 16% formaldehyde</td>
			<td>ThermoFisher Scientific</td>
			<td>28906</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>ThermoFisher Scientific</td>
			<td>QVC0508</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>ThermoFisher Scientific</td>
			<td>16210064</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher Scientific</td>
			<td>28314</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-vGlut1 antibody</td>
			<td>NeuroMab</td>
			<td>75-066</td>
			<td>clone N28/9, 1:100</td>
		</tr>
		<tr>
			<td>Alexa Fluor secondary antibodies</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>17:40</td>
		</tr>
		<tr>
			<td>Spinning disk confocal imaging system</td>
			<td>Andor Technology</td>
			<td>CSU-X1, iXon X3 EMCCD</td>
			<td>60x silicone oil &#38; 20x objectives</td>
		</tr>
	</tbody>
</table>

Source: Nagendran, T., et al., <a href="https://app.jove.com/v/58421">Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons.</a> J. Vis. Exp. (2018)
This video demonstrates immunofluorescence staining of rat hippocampal neurons cultured within a microfluidic chip to analyze neuronal maturation and connectivity.

1. Fluorescence Immunostaining within the Chip

	Prepare 4% formaldehyde fixation solution in PBS (4% formaldehyde, 1 &#181;M MgCl2, 0.1 &#181;m CaCl2, ...

Take a microfluidic chip containing rat hippocampal neurons.
The chip features somatic and axonal reservoirs, with compartments divided by microgrooves, effectively compartmentalizing the neurons and isolating the somatic and axonal regions.
Remove the media and add formaldehyde. Incubate to fix the neurons and preserve their structure.
Remove excess formaldehyde and wash with buffer. Then, add a detergent to permeabilize the neurons.
Replace the detergent with a blocking solution to block non-specific binding sites.
Remove the blocking solution and incubate with primary antibodies that bind to specific neuronal synaptic markers, which are integral membrane proteins on synaptic vesicles.
Remove the unbound antibodies and wash with buffer.
Incubate with fluorophore-tagged secondary antibodies that bind to the primary antibodies targeting synaptic markers.
Remove unbound secondary antibodies and wash with buffer.
Under a confocal microscope, image the chip to visualize the fluorescent neuronal synaptic markers.

11 Views. Immunocolorazione a fluorescenza di neuroni ippocampali di ratto all'interno di un chip microfluidico

Video: Immunocolorazione a fluorescenza di neuroni ippocampali di ratto all'interno di un chip microfluidico - Experiment

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.

Immunocolorazione delle sezioni biocitina-riempite e trasformati per neurochimici Marcatori

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

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Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparazione delle sezioni ricrello della retina

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

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.

Combinazione dell'ibridazione in situ a fluorescenza multiplex con immunoistochimica fluorescente su sezioni cerebrali di topo fresche, congelate o fisse

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

Fluorescence Immunostaining of Rat Hippocampal Neurons within a Microfluidic Chip

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Fluorescence Immunostaining of Rat Hippocampal Neurons within a Microfluidic Chip