Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Summary

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Abstract

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

Introduction

Taste buds are collections of 50-100 specialized epithelial cells that bind subsets of chemical-taste stimuli present in the oral cavity. Taste-transducing cells are generally thought to exist as types^{1,2,3,4,5,6,7,8,9}, initially based on electron microscopy criteria that were later correlated with molecular markers. Type II cells express phospholipase C-beta 2 (PLC^....

Protocol

NOTE: All animals were cared for in accordance with the guidelines set by the U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals and the NIH Guide for the Care and Use of Laboratory Animals. Phox2b-Cre mice (MMRRC strain 034613-UCD, NP91Gsat/Mmcd) or TrkB^CreER mice (Ntrk2^{tm3.1(cre/ERT2)Ddg}) were bred with tdTomato reporter mice (Ai14). Advillin^CreER47 were bred with Phox2b-flpo⁴⁸ and Ai65. For 5-ethynyl-2′-deoxyuridine (EdU) injections, the EdU was prepared and doses calculated according to Perea-Martinez et al.⁴⁹.

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Results

Staining of the lingual epithelium with antibodies to dsRed and keratin-8 (a general taste-bud marker) labeled both whole taste buds and all taste-bud innervation in Phox2b-Cre:tdTomato mice^50,51 (Figure 3A). Imaging these taste buds from their pores to their bases gave the highest resolution x-y plane images (Figure 3A,B). The contour function of the pixel-based imaging program was used.......

Discussion

The development of an approach to consistently collect and stain whole taste buds from three oral cavity taste regions (fungiform, circumvallate, and the palate) provides significant improvements for analyzing taste-transducing cells, tracking newly incorporated cells, innervation, and relationships between these structures. In addition, it facilitates the localization of a potential secondary neuron marker both within or outside of a labeled population⁵⁰. This is particularly relevant given that .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2,2,2-Tribromoethanol	ACROS Organics	AC421430100
2-Methylbutane	ACROS	126470025
AffiniPure Fab Fragment Donkey Anti-Rabbit IgG	Jackson ImmunoResearch	711-007-003	15.5μL/mL
Alexa Fluor® 647 AffiniPure Donkey Anti-Rat IgG	Jackson Immuno Research	712-605-150	(1:500)
AutoQuant X3 software	Media Cybernetics
Blunt End Forceps	Fine Science Tools	FST 91100-12
Click-iT™ Plus EdU Cell Proliferation Kit	Molecular Probes	C10637	Follow kit instructions
Coverglass	Marienfeld	107242
Cytokeratin-8	Developmental Studies Hybridoma Bank (DSHB), (RRID: AB_531826)	Troma1 supernatant	(1:50, store at 4°C)
Dissection Scissors (coarse)	Roboz	RS-5619
Dissection Scissors (fine)	Moria	MC19B
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	ThermoFisher Scientific	A21206	(1:500)
Donkey anti-Rabbit, Alexa Fluor® 555	ThermoFisher Scientific	A31572	(1:500)
DyLight™ 405 AffiniPure Fab Fragment Bovine Anti-Goat IgG	Jackson Immuno Research	805-477-008	(1:500)
Fluoromount G	Southern Biotech	0100-01
Glass slides	Fisher Scientific (Superfrost Plus Miscroscope Slides)	12-550-15
Goat anti-Car4	R&D Systems	AF2414	(1:500)
Imaris	Bitplane	pixel-based image analysis software
Neurolucida 360 + Explorer	MBF Biosciences	3D vector based image analysis software
Normal Donkey Serum	Jackson Immuno Research	017-000-121
Normal Rabbit Serum	Equitech-Bio, Inc	SR30
Olympus FV1000		(multi-Argon laser with wavelengths 458, 488, 515 and additional HeNe lasers emitting 543 and 633)
Paraformaldehyde	EMD	PX0055-3	4% in 0.1M PB
Rabbit anti-dsRed	Living Colors DsRed Polyclonal Antibody; Clontech Clontech Laboratories, Inc. (632496)	632496	(1:500)
Rabbit anti-PLCβ2	Santa Cruz Biotechnology	Cat# sc-206	(1:500)
Sodium Phosphate Dibasic Anhydrous	Fisher Scientific	BP332-500
Sodium Phosphate Monobasic	Fisher Scientific	BP330-500
tert-Amyl alcohol	Aldrich Chemical Company	8.06193
Tissue Molds	Electron Microscopy Sciences	70180
Tissue-Tek® O.C.T. Compound	Sakura	4583
Triton X-100	BIO-RAD	#161-0407
Zenon™ Alexa Fluor™ 555 Rabbit IgG Labeling Kit	ThermoFisher Scientific	Z25305	Follow kit instructions