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

Published: March 29, 2024

Abstract

Source: Mizutani, Y. et al., Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections. J. Vis. Exp. (2018)

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 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 …

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 (1…

開示

The authors have nothing to disclose.

Materials

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
DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) ThermoFisher Scientific D1306
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

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記事を引用
An Immunofluorescence-Based Method for Visualizing Tuft Cells in Jejunum Cryosections. J. Vis. Exp. (Pending Publication), e22102, doi: (2024).

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