An Immunofluorescence-Based Method for Visualizing Tuft Cells in Jejunum Cryosections

Overview

This video illustrates an immunofluorescence-based method for visualizing intestinal tuft cells in free-floating mouse jejunum cryosections. Within the intestinal villi, the co-localization of green fluorescence at the luminal tip with an extended red rootlet mass indicates the presence of tuft cells.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparations

1. Prepare 10 L of phosphate-buffered saline (PBS): 32.27 g of sodium hydrogen phosphate (Na₂HPO₄·12H₂O), 4.5 g of Sodium dihydrogen phosphate (NaH₂PO₄·2H₂O), 80.0 g of sodium chloride (NaCl) added to ultrapure water to a final volume of 10 L. Store solution at room temperature (RT).
2. Prepare 200 mL of PBS-T: 200 mL of PBS from step 1.1 with 100 µL of polyoxyethylene (10) octylphenyl ether to a final concentration of 0.05% (vol: vol). Store at RT.
3. While wearing gloves and eye protection, prepare 50 mL of 15% sucrose in 10% buffered neutral formalin solution: 7.5 g of sucrose added to 10% buffered neutral formalin solution (Table of Materials) to a final volume of 50 mL. Store at RT.
   CAUTION: Inhalation and/or skin/eye contact with formaldehyde in 10% buffered neutral formalin solution can be hazardous. Handle with caution.
4. Prepare 1.5 mL of 200 units/mL phalloidin-fluorescent dye conjugate stock solution: phalloidin-fluorescent dye conjugate (300 units in 1 vial, lyophilized solids, excitation, and emission wavelengths: 581 nm and 609 nm, respectively) suspended in 1.5 mL of methanol. Store the solution in the dark at -20 °C.
5. Prepare 5 mg/mL 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) stock solution: 10 mg of DAPI in 2 mL of N, N-dimethylformamide (DMF). Store the solution in the dark at -20 °C.
6. Prepare 5% bovine serum albumin (BSA) in PBS: 0.5 g of BSA, 10 mL of PBS. Store at 4 °C.
7. Remove the beveled tip of an 18-gauge straight needle using a nipper, and pinch the nipped end with a pincher in a lengthwise direction to allow liquid to flow through the needle lumen (Figure 1A1).
   NOTE: The protocol can be paused here.

2. Animal Dissection and Isolation of Jejunum

1. Perfusion fixation of a mouse
   1. Wear gloves and work in a well-ventilated area.
   2. Prepare a 100 mL glass beaker, 10% buffered neutral formalin solution, surgical tools (scissors, forceps), a metallic tray, 6-0 nylon cut sutures, an 18-gauge straight needle prepared as in 1.7, a 22-gauge winged needle, a 20-mL syringe.
   3. Load 20 mL of 10% buffered neutral formalin solution from the glass beaker into the 20-mL syringe and attach the 22-gauge winged needle to the outlet.
   4. Prepare liquid gelatin by adding 1 g of gelatin powder to 20 mL of PBS (final 5% gelatin) in a 50-mL centrifuge tube. After soaking at RT for 15 min, incubate at 50 °C in a water bath for 15 min without shaking.
   5. Briefly vortex and incubate at 50 °C for another 15 min to completely dissolve the gelatin. Leave the tube at RT until use.
   6. Euthanize an adult mouse by cervical dislocation.
   7. Place the mouse from step 2.1.6 on a metallic tray and make a small incision on the skin through the ensiform cartilage using surgical tools.
   8. Expand the incision with both hands to expose the thoracic and abdominal regions.
   9. Open the peritoneum to expose the diaphragm, and then open the diaphragm on both sides.
   10. Cut the thoracic cage along the anterior axillary lines on both sides, then remove the thoracic wall by cutting in a transverse direction at the position of the thymus to expose the heart.
   11. Make a small incision on the auricle of the right atrium to drain the blood and insert a 22-gauge winged needle into the apex of the left ventricle. Push the plunger to perfuse the tissues with a 10% buffered neutral formalin solution.
   12. After exposing organs in the pelvis, cut off the end of the rectum from the anus, and separate the intestines from the body by cutting the mesentery.
   13. To isolate the jejunum, cut the intestines at 4 cm from the anal side of the gastric antrum. Discard the anal half of the remaining small intestine.
   14. Clip the jejunum in half to facilitate the flushing procedure. Load 20 mL of 10% buffered neutral formalin solution into the 20-mL syringe, and attach the 18-gauge straight needle prepared in step 1.7 to the outlet.
   15. Inject 10% buffered neutral formalin solution from one end of the clipped jejunum to flush out the intestinal contents and to fix the gut lumen surface (Figure 1A2).
   16. Wash the gut lumen by injecting PBS as described in step 2.1.15.
   17. Flush liquid gelatin into the gut lumen as described in step 2.1.15 to replace PBS with liquid gelatin.
   18. Close one end of the clipped jejunum by suture ligation using a 6-0 nylon cut suture, fill the jejunum with liquid gelatin, and close the opposite end by suture ligation (Figure 1A3).
   19. Add four suture knots to fit a sausage-shaped jejunum section to the depressed portion of a cryomold (Figure 1A4).    
       NOTE: For cryomolds with a 20 mm x 25 mm x 5 mm depressed section, a ~20 mm sausage-like jejunum section is preferable.
   20. Soak the tissues in 50 mL of 15% sucrose in 10% buffered neutral formalin solution overnight at 4 °C.        
       NOTE: These steps ensure cryoprotection by sucrose and protein fixation by formalin of gelatin and tissue. Formalin-gelatinized gelatin does not melt at RT, whereas non-fixed gelatinized gelatin at 4 °C melts at RT.

3. Snap Freezing of Gelatin-filled Jejunum Tissues

1. By cutting the jejunum at the suture knots, three sausage-like jejunum pieces with both ends ligated are obtained (Figure 1A4). Align the sausage-like pieces in a cryomold for the addition of embedding compound for the preparation of frozen tissue specimens.
2. Snap freeze the cryomold in isopentane cooled with liquid nitrogen. Store cryomolds at -80 °C 
   NOTE: The protocol can be paused here.

4. Immunofluorescence of Tuft Cells Using Free-floating Cryosections

1. Cryosectioning
   1. Set both the cryostat chamber temperature (CT) and object temperature (OT) to -22 °C. Place the frozen tissue block in a cryomold in the cryostat chamber for at least 15 min.
   2. Add 3 mL of PBS to a 35-mm culture dish.
   3. Remove the frozen tissue block from the cryomold and cut the block in half with a razor blade to expose the transverse section of the jejunum. Mount one half of the block on a chuck (a cryostat adaptor) to section the cutting plane with the razor blade.
   4. Section the gelatin-filled jejunum into 30 µm-thick sections. Use frozen forceps to gently transfer the sections into the 35-mm culture dish prepared in step 4.1.2 (Figure 2B, right).
2. Application of primary antibodies
   1. Wash the free-floating sections in the 35-mm culture dish 3 times for 5 min each with 3 mL PBS-T with mild shaking on a reciprocal shaker (approximately 36 times/min).
   2. Prepare antigen retrieval solution: 0.3 mL of antigen retrieval solution concentrate (Table of Materials) in 2.7 mL of ultrapure water. Add 3 mL of antigen retrieval solution to the 35-mm culture dish containing free-floating sections.
   3. Close the lid, seal the gap between the dish and the lid with a strip of vinyl tape, and incubate at 50 °C in a hybridization incubator for 3 h without shaking.
   4. Remove the dish from the incubator and cool at RT for 20 min. After removing the vinyl tape, wash the sections 3 times for 5 min each with 3 mL of PBS-T and mild shaking.
   5. Aspirate the PBS-T and add 5 drops of blocking solution (Table of Materials) onto the sections. Incubate at RT for 5 min with mild shaking.
   6. Prepare the primary antibody solution: 495 µL of 5% BSA in PBS with 5 µL of phospho-Y1798 girdin (pY1798) antibodies (Table of Materials). Add 500 µL of primary antibody solution onto the sections (the blocking solution does not need to be removed).
   7. Place the dish in a humidified incubation chamber and incubate overnight at 4 °C with mild shaking.       
      NOTE: The overnight incubation can be extended to up to three nights.
3. Application of secondary antibodies
   1. Wash the sections 3 times for 5 min each in 3 mL of PBS-T with mild shaking.
   2. Prepare diluted DAPI stock solution: 2 µL of DAPI stock solution (Step 1.5), 998 µL of PBS.
   3. Make secondary antibody solution: 476 µL of 5% BSA in PBS, 10.5 µL of diluted DAPI solution, 12.5 µL of phalloidin-fluorescent dye conjugate stock solution, 1 µL of goat anti-rabbit IgG-fluorescent dye conjugate (wavelength: excitation 496 nm, emission 520 nm).
   4. After aspirating the PBS-T, apply the secondary antibody solution and incubate in a light-shielded incubation chamber at RT for 30 min with mild shaking.
   5. Wash the sections 3 times for 5 min each with 3 mL of PBS-T and mild shaking.
   6. After the final wash, remove the PBS-T and replace it with 3 mL of PBS lacking polyoxyethylene(10) octylphenyl ether. Transfer the dish to a stereoscopic microscope.
   7. Place 200 µL of PBS in a droplet on the center of a MAS-coated white glass slide and transfer one jejunum section from the dish into the droplet using a P200 pipet tip.
   8. After adjusting the section alignment under the stereoscopic microscope, aspirate all remaining PBS surrounding the section.
   9. Add 20 µL of aqueous mounting media and place a 20 x 20 mm coverslip atop the media.
   10. Immediately seal the coverslip edges with xylene-based mounting media. Place the slide on a wooden mappe and allow the xylene-based mounting media to solidify at RT for 2 - 3 h.     
       NOTE: The protocol can be paused here. After the xylene-based mounting media solidifies, the slides can be stored for 2 - 3 weeks in a light-shielded slide box at RT.

5. Confocal Microscopy

1. Put immersion oil on the 63X objective of a confocal microscope. Turn the coverslip slide down and place the slide on the stage.
2. Digitize the TC images acquired at laser wavelengths 405, 488, and 555 nm and save the images in tiff format (.tiff).     
   NOTE: Choose a detection filter set that is appropriate for the fluorescence dyes (i.e., excitation/emission maxima: 358/461 nm, 490/525, and 590/617).

Representative Results

Figure 1: Gelatin filling of jejunum sections for morphological preservation of cryosections
(A) Photographs of procedures for intraluminal filling of mouse jejunum with gelatin. (A1) The beveled tip of an 18-gauge straight needle was removed to avoid piercing the gut wall. (A2) Buffered neutral formalin solution (10%) was injected in one end of the clipped jejunum to flush the intestinal contents and fix the gut lumen surface. (A3) Clipped jejunum filled with liquid gelatin and both ends ligated using a 6-0 nylon suture (black arrows). (A4) Four more suture knots (white arrows) placed between the pre-existing suture knots (black arrows) yielded three shorter pieces. The tissue will then be separated into three sausage-like pieces at the two indicated positions (white arrowheads). (B) Beneficial effects of gelatin-filling on free-floating cryosection morphology. Without gelatin filling, sections tend to kink, allowing the villi to easily swing backward (left); with gelatin filling, a 30 µm-thick section maintains a disc shape, and the upright position of the villi is preserved (right). Images were photographed using Nomarski differential interference contrast. Scale bars, 1 mm.

Figure 2: Representative fluorescence images of mouse intestinal tuft cells
Confocal fluorescence images of TCs on a villus (A) or in a crypt (B), in free-floating mouse jejunum sections stained with site-specific and phosphorylation-status-specific antibodies against girdin phosphorylated at tyrosine 1798 (pY1798, green, optimal excitation/emission wavelengths 490/525 nm), phalloidin (red, 590/617 nm), 4,6-diamidino-2-phenylindole (DAPI, blue, 358/461 nm). The area enclosed by white boxes in the low magnification images (scale bars, 50 µm) is expanded on the right (scale bars, 10 µm). pY1798 antibodies reproducibly stain TCs, regardless of location (on a villus (A) or in a crypt (B)), with staining present at the lumenal tip (arrows), membrane, and cytoplasm of the spool-shaped TC soma. A prominently thickened brush border in phalloidin staining (arrowheads) is another distinctive sign of TCs. 

Materials

Name	Company	Catalog Number	Comments
Slc:DDY 6-week-old female mice	Chubu Kagaku Shizai	Not applicable
Disodium hydrogenphosphate 12-water (Na2HPO412H2O)	Wako	196-02835
Sodium dihydroenphosphate Dihydrate (NaH2PO42H2O	Wako	199-02825
Sodium chloride (NaCl)	Wako	199-10665
Triton X-100,  Polyoxyethylene(10) octylphenyl ether	Katayama Chemical	Not applicable
Sucrose	Wako	190-00013
10% buffered neutral formalin solution	Muto Chemical	20215	Step 1.3.
Phalloidin-fluorescent dye conjugate, Alexa Fluor 594 phalloidin	ThermoFisher Scientific	A12381
Methanol	Nacalai Tesque	21915-35
N.N-dimethylformamide (DMF)	Nacalai Tesque	13015-75
Bovine serum albumin	Sigma	A9647-10G
18-gauge stainless steel straight needle	Terumo	NN-1838R
U.S.P. 6-0, 60 cm nylon cut suture, Crownjun	Kono Seisakusho	Not applicable
22-gauge winged needle	Terumo	SV-22DLK
20 mL TERUMO syringe	Terumo	SS-20ES
50 mL centrifuge tube	TPP	91050
15 mL Iwaki centrifuge tubes	Iwaki	2325-015
1.5 mL micro test tube	Star	RSV-MTT1.5
Gelatin powder, Gelare-blanc	Nitta Biolab	2809
Embedding compound for frozen tissue specimen, O.C.T. Compound 118 mL 12 piece	Sakura FineTech	4583
Cryomold, CRYO DISH No. 3, 125 piece, well size 20 x 25 x 5 (mm)	Shoei Work's	1101-3
Isopentane	Nacalai Tesque	26404-75
Falcon cell culture dish 35 x 10mm Easy-Grip	Corning	353001
Antigen retrieval solution concentrate, Target Retrieval Solution pH 9 10x	Dako	S2367	Antigen retrieval solution concentrate in step 4.2.2.
Protein Block	Dako	X0909	Blocking solution in 4.2.5.
Phospho-Y1798 girdin (pY1798) antibodies	Immuno-Biological Laboratories (IBL) = Company X	28143	Phospho-Y1798 girdin antibodies in step 4.2.6.
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	ThermoFisher Scientific	A11034
Aquous mounting media, Prolong Gold Antifade Mountant	ThermoFisher Scientific	P36930
Coverslips, Matsunami Micro Cover Glass 22 x 22 mm 100 pcs Thickness No.1	Matsunami Glass	C022221
Entellan New, xylene-based mounting media for microscopy	Merck Millipore	107961
MAS-coated slide glass white	Matsunami Glass	MI-MAS-01
Wooden Mappe KO-type	Shoei Work's	99-40007
Immersion Oil 518 F Fluorescence Free 20 ml	Zeiss	444960
Pipettes, Pipetman P (P2, P20, P200, P1000)	Gilson	F144801, F123600, F123601, F123602
Ultrapure water production system	Advantec	GS-590
Plastic glove, Star Nitrile Glove	Star	RSU-NGVM
Cryostat	Leica Microsystems	CM1950
Deep freezer, SANYO Ultra Low	Sanyo	MDF-382
Showcase refrigerator	Nihon Freezer	NC-ME50EC
Water bath, Thermominder SDminiN (to dissolve gelatin at 50 C)	Taitec	0068750-000
Hybridization incubator (for antigen retrieval at 50 C)	Taitec	HB-100
Incubation chamber for immunostaining	Cosmo Bio	10DO
Reciprocal shaker for immunohistochemistry (for room temperature)	Taitec	NR-1
Reciprocal shaker for immunohistochemistry (for 4 C)	Tokyo Rika Kikai	MMS-3010
Stereoscopic microscope (for tissue handling)	Olympus	SZ61
Stereoscopic microscope (for Figure 1B)	Leica Microsystems	M165FC
Fluorescence microscope (for Figure 3)	Nikon	E800
Confocal laser-scanning microscope	Zeiss	LSM700