Immunofluorescent Staining of Neuronal Populations in the Embryonic Murine Gastrointestinal Tract

Abstract

Source: Barlow-Anacker, A. J., et al., Immunostaining to Visualize Murine Enteric Nervous System Development. 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.

Protocol

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

1. Immunostaining Protocol

1. If samples have been stored in 30% sucrose, rinse them 3 times for 20 min in 1x PBS on a rocking platform.
2. Place the GI tracts into the blocking solution on a rocking platform for 1h at RT.
3. 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 °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).
4. Rinse the GI tracts in 1x PBS 3 times for 5 min and then for 1 hr at RT on a rocking platform.
5. 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 °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 μl of staining solution in order to reduce the volume of antibody used and also to ensure efficient staining of the tissue.
6. Rinse the GI tracts in 1x PBS 3 times for 5 min and then for 1 hr at RT on a rocking platform.
7. 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 °C.
8. Capture images of each fluorophore using a confocal microscope. Use the excitation wavelengths for fluorophores and filters employed in Table 1.
9. Perform computer-aided image analysis using appropriate software depending on the type of confocal used to capture images.

Table 1:  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.

Excitation			Emission
Laser Line	Fluorophore Type	Examples	Photomultiplier Tube	Filter Options
408 nm	Blue	Alexa Fluor 405, Cascade Blue, Coumarin 30, DAPI, Hoechst, Pacific Blue, most quantum dots	1	BP 425-475 nm
488 nm	Green	Alexa Fluor 488, ATTO 488, Calcein, Cy2, eGFP, FITC, Oregon Green, YO-PRO-1	2	BP 500-550 nm
561 nm	Red	Alexa Fluor 546, 555, 568, and 594, Cy3, DiI, DsRed, mCherry, Phycoerythrin (PE), Propidium Iodine (PI), RFP, TAMRA, tdTomato, TRITC	3	BP 570-620 nm
638 nm	Far Red	Alexa Fluor 633 and 647, Allophycocyanin (APC), Cy5, TO-PRO-3	4	BP 663-738 nm

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphate Buffered Saline	Oxoid	BR0014G
Bovine Serum Albumin	Fisher	BP1605
Triton X-100	Sigma	X100
Fluorescence scope	Nikon	SMZ-18 stereoscope
1.5 mL Eppendorf tubes	VWR	20170-038
Fluoromount-G	SouthernBiotech, Birmingham, AL	0100-01
Glass slides	Fisher	12-550-15
Cover glass	VWR	16004-330
Confocal microscope	Nikon	Nikon A1
Nikon Elements	Nikon