immunofluorescent staining of neuronal populations in the embryonic murine gastrointestinal tract

Immunofluorescent Staining of Neuronal Populations in the Embryonic Murine Gastrointestinal Tract

Place the GI tracts into a 0.2-milliliter tube containing blocking solution, consisting of 1x PBS, 3% bovine serum albumin, 0.1 Triton X-100, and rock for 1 hour at room temperature. After blocking, aspirate the blocking solution with a fine-tipped Pasteur pipette and replace with the optimal dilution of primary antibodies diluted in blocking solution.
Incubate for either four hours at room temperature or overnight at 4 degrees Celsius on the rocking platform. Following the primary antibody incubation, aspirate the antibody solution and rinse the GI tracts in PBS three times for five minutes each, and then for one hour at room temperature with rocking.
After the last wash, replace the PBS with fluorophore-conjugated secondary antibodies diluted in blocking solution, and place the tubes on the rocking platform in the dark for four hours at room temperature, or overnight at 4 degrees Celsius.
After washing the GI tracts as before, place a few drops of fluorescence mounting media with DAPI onto a glass slide, then, immerse the GI tract into the mounting medium and add a cover glass. Finally, capture images of each fluorophore using a confocal microscope, then perform computer-aided image analysis using appropriate software.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunostaining Protocol
<ol>
	<li>If samples have been stored in 30% sucrose, rinse them 3 times for 20 min in 1x PBS on a rocking platform.</li>
	<li>Place the GI tracts into the blocking solution on a rocking platform for 1h at RT.</li>
	<li>Remove the blocking solution and incubate the GI tracts with the appropriate amount of primary antibodies diluted in the blocking solution for either 4 hr at RT or O/N at 4 &#176;C on a rocking platform. Use a 1:1,000 dilution of human anti-Hu antibody (serum obtained from the patient), a 1:1,000 dilution of chicken anti-green fluorescent protein (GFP) antibody, and a 1:100 dilution of goat anti-ChAT antibody. 
	NOTE: We utilize a human anti-Hu antibody that was obtained locally from a patient; however, anti-Hu antibodies are commercially available, for example, mouse anti-Hu (use at 1:500 dilution).</li>
	<li>Rinse the GI tracts in 1x PBS 3 times for 5 min and then for 1 hr at RT on a rocking platform.</li>
	<li>Replace the 1x PBS with secondary antibodies diluted 1:500 in blocking solution on a rocking platform for either 4 hr at RT or O/N at 4 &#176;C. Use donkey anti-human amine-reactive dye such as Dylight, donkey anti-chicken Cy2 and donkey anti-goat Cy5. If using the mouse anti-Hu antibody, then use a 1:500 dilution of donkey anti-mouse amine-reactive dye 405. 
	NOTE: The intensity of the endogenous GFP and tdTomato expression and the clarity of immunostaining increase with developmental age. We typically immunostain embryonic guts individually in 0.2 ml tubes with 150 &#956;l of staining solution in order to reduce the volume of antibody used and also to ensure efficient staining of the tissue.</li>
	<li>Rinse the GI tracts in 1x PBS 3 times for 5 min and then for 1 hr at RT on a rocking platform.</li>
	<li>Place a few drops of fluorescence mounting medium with DAPI onto a glass slide. Immerse the GI tract into the fluorescence mounting medium and add a cover glass directly on top of the tissue. The fluorescence mounting medium -G is a water-soluble compound that provides a semi-permanent seal. 
	NOTE: Use fluorescence mounting medium such as Vectashield, a glycerol-based mounting medium that prevents antibody fading and photobleaching. Both of these products enable tissues to be stored for long periods of time at 4 &#176;C.</li>
	<li>Capture images of each fluorophore using a confocal microscope. Use the excitation wavelengths for fluorophores and filters employed in Table 1.</li>
	<li>Perform computer-aided image analysis using appropriate software depending on the type of confocal used to capture images.</li>
</ol>
Table 1: &#160;Confocal imaging excitation wavelengths, fluorophores, and filters. The excitation wavelengths, detectable fluorophores, and filters employed are presented. This information can be used in choosing combinations of secondary antibodies for multicolor immunofluoresence.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Excitation&#160;&#160;&#160; </td>
			<td colspan="2">Emission</td>
		</tr>
		<tr>
			<td>Laser Line</td>
			<td>Fluorophore Type</td>
			<td>Examples</td>
			<td>Photomultiplier Tube</td>
			<td>Filter Options</td>
		</tr>
		<tr>
			<td>408 nm</td>
			<td>Blue</td>
			<td>Alexa Fluor 405, Cascade Blue, Coumarin 30, DAPI, Hoechst, Pacific Blue, most quantum dots</td>
			<td>1</td>
			<td>BP 425-475 nm</td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>Green</td>
			<td>Alexa Fluor 488, ATTO 488, Calcein, Cy2, eGFP, FITC, Oregon Green, YO-PRO-1</td>
			<td>2</td>
			<td>BP 500-550 nm</td>
		</tr>
		<tr>
			<td>561 nm</td>
			<td>Red</td>
			<td>Alexa Fluor 546, 555, 568, and 594, Cy3, DiI, DsRed, mCherry, Phycoerythrin (PE), Propidium Iodine (PI), RFP, TAMRA, tdTomato, TRITC</td>
			<td>3</td>
			<td>BP 570-620 nm</td>
		</tr>
		<tr>
			<td>638 nm</td>
			<td>Far Red</td>
			<td>Alexa Fluor 633 and 647, Allophycocyanin (APC), Cy5, TO-PRO-3</td>
			<td>4</td>
			<td>BP 663-738 nm</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Oxoid</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>BP1605</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence scope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech, Birmingham, AL</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>16004-330</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>Nikon A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Elements</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Barlow-Anacker, A. J., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52716">Immunostaining to Visualize Murine Enteric Nervous System Development.</a> J. Vis. Exp. (2015)
This video demonstrates the immunostaining of the embryonic murine gastrointestinal tract to visualize and analyze the neuronal population distribution using immunofluorescence and confocal microscopy.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunostaining Protocol

	...

Take a fixed, transgenic embryonic mouse gastrointestinal tract at the desired developmental stage.
The gastrointestinal tract&#39;s enteric nervous system includes enteric neurons and cholinergic neurons expressing choline acetyltransferase.
These cholinergic neurons also express a fluorescent protein driven by the choline acetyltransferase promoter.
Incubate the tract with a non-ionic detergent and proteins to permeabilize cells and prevent non-specific antibody binding.
Replace with primary antibodies. Incubate for the antibodies to bind their target proteins in neurons, respectively.
Remove the unbound antibodies and wash with buffer.
Incubate with fluorophore-conjugated secondary antibodies to specifically bind the primary antibodies bound to target proteins.
Remove the excess antibodies and wash with buffer.
Immerse the labeled tract in mounting media containing a nuclear dye to stain the nuclei. Now, mount a coverslip.
Using confocal microscopy, visualize and analyze the distribution of cholinergic neurons in the gastrointestinal tract.

14 Views. Immunofluorescent Staining of Neuronal Populations in the Embryonic Murine Gastrointestinal Tract

Video: Immunofluorescent Staining of Neuronal Populations in the Embryonic Murine Gastrointestinal Tract - Experiment

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JoVE Journal

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Immunofluorescent Staining of Neuronal Populations in the Embryonic Murine Gastrointestinal Tract

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Immunofluorescent Staining of Neuronal Populations in the Embryonic Murine Gastrointestinal Tract