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ProtocolIsolation of myenteric and submucosal plexus
from mouse gastrointestinal tract andsubsequent flow cytometry andimmunofluorescence
Tomasz Ahrends,
Molly Weiner, Daniel
Mucidatahrends@rockefeller.edu(T.A.)
mucida@rockefeller.edu(D.M.)

Highlights
We describe themethod for physicalseparation of mouseintestinal layers
Flow cytometry
analysis of immune
cell subsets inmyenteric andsubmucosal plexus
Immunofluorescenceanalysis of entericnervous system inmyenteric andsubmucosal plexus

The myenteric plexus is located between the longitudinal and circular layers of muscularis externain the gastrointestinal tract. It contains a large network of enteric neurons that form the entericnervous system (ENS) and control intestinal functions, such as motility and nutrient sensing. This
protocol describes the method for physical separation (peeling) of muscularis and submucosal
layers of the mouse intestine. Subsequently, the intestinal layers are then processed for flow
cytometry and/or immunofluorescence analysis.
Ahrends et al., STAR Protocols
3, 101157
March 18, 2022 ª 2022 The
Authors.
https://doi.org/10.1016/
j.xpro.2022.101157

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OPEN ACCESS

ProtocolIsolation of myenteric and submucosal plexus frommouse gastrointestinal tract and subsequent flow
cytometry and immunofluorescence
Tomasz Ahrends,1,3,* Molly Weiner,1 and Daniel Mucida1,2,4,*
1Laboratory of Mucosal Immunology, The Rockefeller University, New York, NY, USA
2Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA
3Technical contact
4Lead contact*Correspondence: tahrends@rockefeller.edu (T.A.), mucida@rockefeller.edu (D.M.)
https://doi.org/10.1016/j.xpro.2022.101157

SUMMARY

The myenteric plexus is located between the longitudinal and circular layers ofmuscularis externa in the gastrointestinal tract. It contains a large network ofenteric neurons that form the enteric nervous system (ENS) and control intestinal
functions, such as motility and nutrient sensing. This protocol describes themethod for physical separation (peeling) of muscularis and submucosal layersof the mouse intestine. Subsequently, the intestinal layers are then processed
for flow cytometry and/or immunofluorescence analysis.
For complete details on the use and execution of this profile, please refer to Ahrends et al. (2021).
BEFORE YOU BEGIN
The protocol below describes the specific steps for the isolation and analysis of mouse distal small
intestine (ileum). However, we have also used this exact protocol for proximal small intestine (duodenum) and large intestine (colon). The protocol focuses on the analysis of steady-state myentericplexus, but we have also used it to describe changes in enteric neuron numbers and muscularis
macrophage phenotype following enteric infection and microbiome manipulation.
Animal studies must be approved by an Institutional Animal Care and Use Committee (IACUC) andperformed in accordance with IACUC guidelines.
Mice
Mouse strain selection will depend on the experiment. We have extensively analyzed 7–12 week old
C57BL/6J mice, as well as various transgenic strains.
Prepare Sylgard plates
Timing: 48 h
Sylgard-coated plates are used to pin the intestinal layers for fixation following their separation.
1. Mix 10 parts Sylgard Base with 1 part Sylgard Curing Agent.
2. Add 2 g of Charcoal Powder and mix thoroughly with a tongue depressor.
3. Pour into Single Well plate, fill 1/3 of the well depth and set in a level, dust-free location to solidify
for 48 h at 20 C–25 C.

STAR Protocols 3, 101157, March 18, 2022 ª 2022 The Authors.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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ProtocolOPEN ACCESSKEY RESOURCES TABLEREAGENT or RESOURCESOURCEIDENTIFIER

Antibodies
CD64-APC (Clone X54-5/7.1)

BioLegendcat. # 13906

CD11b-FITC (Clone M1/70)

BD

cat. # 553310

CD206-AF700 (Clone MMR)

BioLegendcat. # 141732

Arg1-PE-Cy7 (Clone A1exF5)

Thermo Fisher Scientificcat. # 25-36-97-82

CD45.2-PerCP-Cy5.5 (Clone 104)

BD

cat. # 552950

Human polyclonal anti-Hu (ANNA-1)

Gift of V. Lennon

N/A

Goat anti-human IgG (H+L)-AF568

Thermo Fisher Scientificcat. # A-21090

Chemicals, peptides, and recombinant proteins
LIVE/DEAD Fixable Near-IR Cell Stain Kit

Thermo Fisher Scientificcat. # L34975

HBSS, calcium, magnesium

GIBCO

cat. # 24020117

Dulbecco’s Phosphate Buffered Saline (PBS)

Corning

cat. # 21040CV

Fetal Bovine Serum (FBS)

Sigma-Aldrichcat. # F0926-500ML

Collagenase D

Thermo Fisher Scientificcat. # 50-100-3282

DispaseCorning

cat. # CB40235

HEPES 1M

Invitrogencat. # 15630-080

Triton X-100

Alfa Aesar

cat. # A16046

Normal Goat Serum

Jackson ImmunoResearchcat. # 005-000-121

Fluoromount-G

SouthernBiotech

cat. # 0100-01

16% Paraformaldehyde (PFA)

Electron Microscopy Sciences

Cat. # 15714-S

Sodium Pyruvate (NaPyr) (100mM)

Invitrogencat. # 11360070

Bovine Serum Albumin (BSA)

Jackson ImmunoResearchCat. # 001-000-162

Sylgard 184 Silicone Elastomer

Electron Microscopy Sciences

Cat. # 24236-10

Activated charcoal powder

Sigma-AldrichCat. # 05105-250G

DNase I

Roche

cat. # 04536282001

Thermo Fisher Scientificcat. # 00-5523-00

The Jackson Laboratory#000664

Fiji

ImageJ

https://imagej.net

FlowJoTree Star

RRID:SCR_008520

Microscope Slides Fisherbrand Superfrost Plus White

Fisher ScientificCat. # 12-550-15

Rectangular Coverslips 40 3 22mm

Thermo Fisher ScientificCat. # 2980-224

Tongue Depressors

Thermo Fisher Scientificcat. # 23-400-121

Minutien Pins, 0.22mm diameter

Roboz Surgical Instruments

cat. # RS-6083-20

Nunc OmniTray Single-Well Plates with Lids

Thermo ScientificCat. # 242811

Critical commercial assays
eBioscience Foxp3 / Transcription Factor Staining Buffer Set
Experimental models: Organisms/strains
Mouse: C57BL6/J
Software and algorithms

Other

MATERIALS AND EQUIPMENT
Digestion Buffer
Reagent

Final concentration

HBSS Ma2+/Ca2+ + 5% FCS

Amount
7.6 mL

Collagenase D

400 U/mL

Dispase

2.5 U/mL

500 mL

HEPES

25 mM

250 mL

NaPyr

1 mM

100 mL

DNase I50 mg/mL

50 mL

Total

10 mL

Store at 4 C for on ice and use on the same day.

2

1 mL

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ProtocolOPEN ACCESSFACS Buffer
Reagent

Final concentration

Amount

FCS

2%

1 mL

PBS

49 mL

Total

50 mL

Store at 4 C for up to 1 week.

4% Paraformaldehyde (PFA) solution
Reagent

Final concentration

Amount

16% PFA solution

4%

12.5 mL

PBS

37.5 mL

Total

50 mL

Store at 4 C up to 1 month.

Permeabilization Buffer
Reagent

Final concentration

Amount

Triton-X

0.2%

2 mL

PBS

998 mL

Total

1000 mLStore at 20 C–25 C for up to 1 year.

Blocking Buffer
Reagent

Final concentration

Permeabilization Buffer
BSA (Bovine Serum Albumin)

5%

Normal Goat Serum

5%

Total

Amount45.2 mL
2.4 g
2.4 mL
50 mL

Store at 4 C for up to 2 weeks.

Note: Depending on the antibodies used for immunofluorescent staining, the source of serum
should be adjusted – e.g., if secondary donkey antibodies are used, prepare blocking buffer
with normal donkey serum.

STEP-BY-STEP METHOD DETAILS
Separation of mouse intestinal layers
Timing: 15 min
1. Separation of myenteric and submucosal plexus.
a. Euthanize mouse by cervical dislocation or other approved method.
b. Lay euthanized mouse on its back on a cold surface (e.g., metal dissection block placed on ice);spray abdominal area with 70% ethanol. Lift the skin of the lower abdomen with tweezers andmake a V-shaped incision with scissors; cut away skin to expose abdominal cavity.
c. Locate the beginning of the small intestine at the base of the stomach; cut the beginning of thesmall intestine from the stomach and carefully pull out from abdominal cavity, allowing mesenteric fat to peel away from intestine as it is pulled, and gently removing as much remaining fat
as possible with tweezers.

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d. Divide the small intestine into four equal pieces; collect the ileum, which is the most distal
fourth, approximately 6–7 cm of small intestinal tissue proximal to the beginning of thececum).
Note: To account for variation in neuronal density in different intestinal segments, analysis
should always be performed with the same part of the small intestine (e.g., last 3 cm of
the ileum).
e. Insert scissors into intestinal opening; carefully pull intestine over scissor blade; make astraight cut to the right of the mesenteric attachment over the length of the tissue until intestine is fully opened (pulling the entire length of tissue onto the scissor blade allows fewer cutsand enables a straighter cut, which prevents tearing of the tissue during the layer separation).
f. Locate the outside layer of the intestine (the smooth side with mesenteric attachment, as seen
in Methods video S1 at 00:30 min and Figure 1); grip with tweezers and vigorously shake in apetri dish filled with PBS on ice to wash.
g. Place the intestine on a cold surface (e.g., metal dissection block) with the muscularis (outer)layer facing up (luminal side facing down). Use small, damp paint brushes to completely flatten
the tissue, making sure the sides don’t fold under (See Methods video S1 at 00:06 min).
h. With one pair of tweezers, grip the intestine at the beginning of the tissue segment, and use asecond pair to gently make a dent in the tissue directly below the pair of gripping tweezers,
making sure not to pierce through the entire thickness of the tissue (as seen in Methods video
S1 at 00:45 min); carefully stroke downwards on the tissue from the indentation with second
pair of tweezers until you see the layers separating, and the muscularis rolling away. Gradually
adjusting both tweezers down the length of the tissue, keep scraping the muscularis layeraway until you reach the end of the length tissue. See Methods video S1.
Note: Depending on the experimental question, submucosal layer (tissue remaining after
muscularis isolation) of the intestine can be similarly analyzed by flow cytometry or
microscopy.
i. Store tissue in PBS on ice until all animals are processed.
2. Depending on the desired analysis, immediately continue with ‘‘flow cytometry preparation’’ or‘‘immunofluorescence preparation’’.
Note: Flow cytometry and immunofluorescent analysis can be performed together for eachsample. Use 1/3 of the tissue for imagining and 2/3 for flow cytometry. Make sure to use
the same part of the tissue for each type of analysis (e.g., lower ileum for imaging and upper
ileum for flow cytometry).

Immunofluorescence preparation
Timing: 4 days
3. Place tissue on a Sylgard-coated plate; stretch and flatten using damp paint brushes, avoiding
any wrinkles or folds, and pin down with Minutien pins. See Figure 2.
4. Cover the pinned tissue with 4% PFA solution and keep at 4 C for 16 h.
5. Discard PFA solution and transfer tissue to a 24-well plate filled with PBS. Wash 4 times for 15 min
per wash at 20 C–25 C on an orbital shaker, transferring tissue to a fresh well of PBS after eachwash.
6. Transfer tissue to 24-well plate filled with Permeabilization Buffer. Incubate at 20 C–25 C on anorbital shaker for 2 h, moving the tissue to a second PBS-filled well after 1 h.

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Figure 1. Peyer’s patches and mesenteric attachment visible on the muscularis layer of the mouse small intestine

Pause Point: Tissue can be kept in Permeabilization Buffer at 4 C for up to 1 week. For longer
storage (2 months) secure the plate with parafilm and keep tissue in Permeabilization Buffer
supplemented with 0.05% sodium azide.
7. Transfer tissue to 24-well plate filled with Blocking Buffer. Incubate at 20 C–25 C on an orbital
shaker for 1 h.
8. Prepare 600 mL of primary antibody solution per 4 cm piece of muscularis, diluted in Blocking
Buffer. Place tissue in antibody solution in 24-well plate; seal with parafilm and incubate for
48 h at 4 C on a platform rocker.
9. Transfer tissue to a 24-well plate filled with PBS. Wash 4 times for 15 min per wash at 20 C–25 C
on an orbital shaker, transferring tissue to a new PBS-filled well after each wash.
10. Prepare 600 mL of secondary antibody solution per 4 cm piece of muscularis, diluted in Permeabilization Buffer. Incubate tissue in solution in a 24-well plate for 2–3 h at 20 C–25 C, coveredfrom light on an orbital shaker.
11. Transfer tissue to a 24-well plate filled with PBS. Wash 4 times for 15 min per wash at 20 C–25 C
on an orbital shaker, transferring tissue to a new PBS-filled well after each wash.
12. Transfer tissue to a microscope slide, completely flatten with damp paint brushes, cover with afew drops of Fluoromount-G mounting medium and carefully place a coverslip on the tissue
sample. Avoid introducing air bubbles by lowering the coverslip gradually from one side to
the other.
13. Allow slides to dry for 16 h in dark at 20 C–25 C, or until the media solidifies.
Optional: For long-term storage (> 4 weeks) the slides can be sealed with nail polish.
Pause Point: Stained slides may be stored in the dark at 4 C for at least 2 weeks prior to microscopy analysis.
14. Acquire 10 randomly selected, evenly spaced, complete z-stacks for each slide.
For our studies, we used a Zeiss LSM 780 confocal microscope with 253 glycerol objective, 0.63
zoom, 1 airy unit pinhole and 512 3 512 pixels xy resolution.

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Figure 2. Sylgard plate with multiple pinned fragments of ileum muscularis

Flow cytometry preparation
Timing: 3–4 h
15. If analyzing the submucosa, remove Peyer’s patches with scissors or tweezers (there are 6–10
visible Peyer’s patches in the entire small intestine). See Figure 1.
16. Place tissue into a 6-well plate without liquid and use scissors to cut thoroughly into very fine(less than 0.5 mm) pieces, for approximately 15 s.
17. Add Digestion Buffer to the plate and place it on a shaker at 37 C. Use following times andDigestion Buffer volumes (longer digestion time is required for muscularis to ensure properdissociation):a. Muscularis: 40 min in 4 mL.
b. Mucosae: 20 min in 6 mL.
18. Following digestion, homogenize tissue with 18G needle/10 mL syringe by aspirating up anddown twice.
19. Add 5 mL HBSS+5% FCS to each well.
20. With a 10 mL serological pipet, filter the cell suspension through a 70 mm cell strainer directly
into a 15 mL falcon tube. If 5–6 cm of tissue is analyzed, approximately 6 3 106 cells are recovered from muscularis and 10 3 106 from the submucosal layer.
21. Centrifuge at 400 g for 5 min at 4 C, aspirate supernatant, resuspend the pellet in 100 mL of
FACS buffer and pipette to transfer the cells to a round-bottom 96-well plate.
22. Centrifuge plate at 400 g for 5 min at 4 C and remove supernatant by quickly inverting the plateover the sink.
23. Resuspend cells in 50 mL of surface antibody mix, diluted in FACS buffer. Incubate for 30 min on
ice, covered from light.
24. Wash cells by adding 100 mL of FACS buffer to each well. Centrifuge plate at 400 g for 5 min at
4 C and remove supernatant by quickly inverting the plate over the sink.
Optional: For intracellular staining, make Fix/Perm Buffer by diluting 1 part Fix/Perm Concentrate in 3 parts Fix/Perm Diluent from the Fixation/Permeabilization Solution Kit (See key resources table). Resuspend cells in 50 mL of Fix/Perm Buffer and incubate for 30 min on ice,and covered from light. Wash cells by adding 100 mL of Perm buffer to each well. Centrifuge
at 400 g for 5 min at 4 C and remove supernatant by quickly inverting the plate over the sink.

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Figure 3. Neuronal quantification in the ileum myenteric and submucosal plexus assessed by IF staining of ANNA-1
Left: representative image; scale bar 50 mm

Resuspend cells in 50 mL of intracellular antibody mix, diluted in Perm buffer (diluted 1:10 inwater). Wash cells by adding 100 mL of Perm buffer to each well. Centrifuge at 400 g for 5 min
at 4 C and remove supernatant by quickly inverting the plate over the sink.
25. Resuspend cells in 300 mL of FACS buffer and transfer to FACS tubes.
26. Acquire samples on FACS machine.
a. For our studies, we used LSR-II flow cytometer (Becton Dickinson, USA) and analyzed the datausing FlowJo software package (Tri-Star, USA).

EXPECTED OUTCOMES
For a sample image of immunofluorescence analysis see Figure 3. For sample plots of flow cytometry
analysis see Figure 4.

QUANTIFICATION AND STATISTICAL ANALYSIS
Immunofluorescence image analysis was performed in Fiji. The ‘‘Cell counter’’ plugin was used toenumerate neuronal cell bodies in each image. For each sample, 10 randomly selected images
were acquired and average number of neurons per sample was calculated. To calculate the densityof neurons per mm2, the average number of neurons in each sample was multiplied by 3.125. This
number was calculated based on the dimensions of acquired images and microscope magnification
settings.
For representative gating strategy of flow cytometry data see Figure 4.

LIMITATIONS
Depending on mouse strain, age, treatment conditions, and intestinal segments, we observed variability of the thickness of each layer. Thin muscularis layer (e.g., in Balb/c mice, weanlings, or in the
duodenum) is prone to tearing during the separation.

TROUBLESHOOTING
Problem 1
Thin muscularis layer (See step 1).
Potential solution
For immunofluorescence analysis, cut open the intestine and fix the tissue without separating thelayers, then separate after fixation. This makes the muscularis layer stiffer and therefore easier toisolate. If this method is used, all samples should be processed in this way to avoid technicalSTAR Protocols 3, 101157, March 18, 2022

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Figure 4. Flow cytometry analysis of macrophages isolated from ileum muscularis

variability. Flow cytometry will not be possible if samples are fixed prior to single-cell suspension
preparation.
Problem 2
Weak staining/signal in immunofluorescence analysis. (See step 14)
Potential solution
Samples can be re-stained. Submerge each slide in PBS for 5 h (or 16 h) to dissolve the mounting
media and release tissue samples. Continue with a regular staining protocol as outlined above(see step 6).
Problem 3
Thin muscularis layer in weanlings. (See step 1)
Potential solution
Immunofluorescence analysis of enteric neurons in weanlings (up to 3 weeks old) can be performed
without the separation of the layers. Due to low thickness of the tissue, both layers can be imaged by
confocal microscopy.
Problem 4
Low cell viability during flow cytometry analysis. (See step 26)

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Potential solution
Limit the time between the separation of intestinal layers and enzymatic digestion, ideally to lessthan 1 h. If one person is performing the experiment, up to 10 mice should be analyzed at a time.
Try staggering experimental groups in time or dividing the workload between two people.
Problem 5
Inconsistent enteric neuron densities after image analysis (see step 14). This can occur when small
intestine stretches due to experimental manipulations e.g., long-term antibiotic treatment.
Potential solution
Instead of density per mm2, the quantification of enteric neurons can be performed per ganglion. A
myenteric ganglion can be defined as a continuous group of ANNA1+ cells (neurons) that are separated by less than 15 mm in distance. Only complete ganglia should be counted per field of view. The
following ganglia should be excluded: 1. Ganglia that were truncated; 2. No clear separation (>15 mm) was noted between the last ANNA1+ cell and the edge of the field of view. In the case of
single ANNA1+ cells that are separated by 15 mm on all sides, this was counted as extraganglionic.
The number of quantifiable ganglia was averaged across a minimum of 10 images per gut segment
per animal.

RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Daniel Mucida (mucida@rockefeller.edu).
Materials availability
This study did not generate new unique reagents.
Data and code availability
This study did not generate/analyze datasets/code.

SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.xpro.2022.101157.

ACKNOWLEDGMENTS
This work was supported by NIH Transformative (R01DK116646 and R01DK126407), Kenneth Rainin
Foundation, and Food Allergy FARE/FASI Consortium to D.M. T.A. is a Human Frontier of Science
Program postdoctoral fellow.

AUTHOR CONTRIBUTIONS
Methodology, T.A. and M.W.; Writing, T.A.; Editing, M.W. and D.M.; Funding and supervision, D.M.
All authors critically reviewed, edited, and approved the final manuscript.

DECLARATION OF INTERESTS
The authors declare no competing interests.

REFERENCES
Ahrends, T., Aydin, B., Matheis, F., Classon,
C.H., Marchildon, F., Furtado, G.C., Lira, S.A.,

and Mucida, D. (2021). Enteric pathogens
induce tissue tolerance and prevent neuronalloss from subsequent infections. Cell 184, 5715–
5727.

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