All experiments and procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the committee for Laboratory Animal Care and Use at Kyushu University, Japan (approval no. A19-003).
The experimental subjects were adult male and female C57BL/6NCrj (B6) mice (age, 8–16 weeks; weight, 20–32 g; Charles River, Tokyo, Japan) and their littermates (postnatal day 4–8) as well as transgenic T1R3-GFP mice co-expressing taste receptor type 1 member 3 (T1R3) and green fluorescent protein (GFP)33. All mice were housed in a constant room temperature of 24 ± 1 °C under a 12-hour light and 12-hour dark cycle (lights on at 0800) and given access to food and water ad libitum.
DNA microarray analysis
Taste tissues and cranial ganglia were collected from B6 mice under pentobarbital anesthesia (50–60 mg/kg body weight). The trachea of each mouse was cannulated, and then the mouse was fixed in the supine position with the head in a holder. The GG, NPG and TG were dissected. Mouse individual taste buds were isolated from FP and CV in the peeled tongue epithelium by aspiration with a transfer pipette. Total RNAs were purified from the collected tissue samples using the RNeasy Plus Micro kit (Qiagen, Stanford, CA, USA). The GeneChip Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA, USA) was used for microarray analysis. RNA quality control, total RNA labeling, microarray hybridization and scanning were performed in accordance with the Affymetrix GeneChip Expression Analysis Technical Manual (www.affymetrix.com).
RT-PCR was conducted as described previously34,35,36. Taste buds were isolated from the FP and CV of each mouse by using a transfer pipette, and pooled. The RNeasy Plus Micro kit (Qiagen) was used to purify RNAs from 30 taste buds from the FP and CV respectively, or a 1 mm × 1 mm block of epithelial tissue without taste buds. cDNAs were generated by RT [oligo(dT)12–18 primer] with the superscript pre-amplification system (Invitrogen, Carlsbad, CA, USA). The primer sequences are shown in Table 2. To control for signals from genomic DNA, purified RNA samples were treated in parallel with or without reverse transcriptase. PCR reaction was carried out under the following condition: 95 °C for 15 min (1 cycle); 94 °C for 30 s, 55 °C for 30 s and 72 °C for 36 s (35 cycles). Each 20 μL of PCR solution was comprised of 0.5 U of TaqDNA polymerase (TaKaRa Ex TaqHS; Takara Bio, Kusatsu, Japan), 2 μL of 10× PCR buffer containing 20 mM Mg2+, 0.2 mM of each dNTP and 0.6 μM of each primer pair. The resulting amplification products were visualized in a 2% agarose gel with 0.5 μg/mL ethidium bromide. β-actin was used as the internal control.
RNA probes were prepared for ISH as previously described30,34,37,38. Primer sequences for ISH are listed in Table 2. RT-PCR products were purified and cloned into the pGEM T-Easy vector (Promega, Madison, WI, USA), confirmed by DNA sequencing and digested with appropriate restriction enzymes. Digoxigenin-UTP-labeled antisense and sense RNA probes were prepared by in vitro transcription using the SP6/T7 RNA polymerase Kit (Roche, Mannheim, Germany). Frozen blocks of the dissected tongue and cranial ganglia of B6 mice were embedded in optimal cutting temperature (OCT) compound (Sakura Finetechnical, Tokyo, Japan) and sliced into serial sections (6-μm thickness), which were placed on silane-coated glass slides. The cryosections were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 min at room temperature, washed with 5× standard saline citrate (SSC) for 15 min at room temperature, incubated in prehybridization solution (5 × SSC/50% formamide) for 1 h at room temperature, and then hybridized in a hybridization buffer containing 50% formamide, 5 × SSC, 5 × Denhardt’s solution, 250 μg/mL denatured baker’s yeast tRNA, 500 μg/mL denatured salmon testis DNA, 1 mM dithiothreitol and 20–200 ng/mL antisense RNA probe for 18 h at 60 °C. Subsequently, the sections were washed with 5 × SSC/50% formamide two times for 5 min and with 0.2 × SSC/50% formamide two times for 60 min at 65 °C. The sections were immersed in Tris-buffered saline (TBS) consisting of 50 mM Tris/HCl (pH 7.5) and 150 mM NaCl for 5 min at room temperature, then incubated with blocking solution containing 1% blocking reagent (Roche) in TBS for 60 min, and reacted with anti-digoxigenin Fab fragments conjugated with alkaline phosphatase (1:400 dilution; Roche) in blocking solution for 60 min at room temperature. After washing three times for 5 min with Tris-NaCl-Tween (TNT) buffer containing 50 mM Tris/HCl (pH 7.5), 150 mM NaCl and 0.05% Tween 20, the sections were rinsed in buffer comprising of 100 mM Tris/HCl (pH 9.5), 100 mM NaCl and 50 mM MgCl2 for 5 min. The signals were developed using 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium chloride as chromogenic substrates. The slides were rinsed in Tris-EDTA buffer to stop the reaction and then mounted. The signal specificity of the mRNA for each gene was investigated in parallel experiments using the corresponding sense probes as a negative control.
After the ISH procedure, the sections were washed with TNT buffer three times for 5 min, then preincubated with 1% blocking reagent (Roche) for 1 h at room temperature, and reacted with primary antibody against T1R3 (1:100; goat anti-T1R3, cat. no. sc-22458, Santa Cruz Biotechnology, Dallas, TX, USA), gustducin (1:100; rabbit anti-Gαgust(I-20), cat. no. sc-395, Santa Cruz Biotechnology) or Car4 (1:100; goat anti-CA4, cat. no. AF2414, R&D Systems, Minneapolis, MN, USA) in blocking reagent overnight at 4 °C. The sections were washed with TNT buffer three times for 5 min, and then reacted with appropriate secondary antibody in 1% blocking reagent: Alexa Fluor 568 donkey anti-goat IgG (Invitrogen) for T1R3, Alexa Fluor 555 donkey anti-rabbit IgG (Invitrogen) for gustducin and Alexa Fluor 568 donkey anti-goat IgG (Invitrogen) for Car4 for 2 h at room temperature. The images of the labeled taste cells were taken using the FV1000 confocal laser scanning microscope and Fluoview software (Olympus, Tokyo, Japan). We counted positively-staining cells in each taste bud in horizontal sections of the FP and CV. To exclude artifactual signals, Image-Pro Plus 4.0 (Media Cybernetics, Rockville, MD, USA) was used, that is cells showing a signal density greater than the mean plus two standard deviations of the signal density of taste cells in the negative control (primary antibody omitted) were considered positive. The same positive cells observed on contiguous sections were counted only once.
For immunohistochemical detection of Pcdh20 protein in T1R3-GFP mice, dissected tongues were fixed in 4% PFA in PBS for 45 min at 4 °C, and dehydrated with sucrose solutions (10% for 1 h, 20% for 1 h and 30% for 3 h, at 4 °C). The tongue frozen block embedded in OCT compound (Sakura Finetechnical) was sliced into serial sections (8-μm thickness), placed on silane-coated glass slides and air dried. After washing with TNT buffer, the sections were immersed in 1% blocking reagent (Roche) for 1 h at room temperature, and reacted with primary antibody against Pcdh20 (1:100; rabbit anti-Pcdh20, cat. no. bs-11113R, Bioss Inc., MA, USA) in blocking reagent overnight at 4 °C. After washing with TNT buffer three times for 5 min, sections were reacted with Alexa Fluor 647 donkey anti-rabbit IgG secondary antibody (Invitrogen) in 1% blocking reagent for 2 h at room temperature. The sections were washed with TNT buffer. The immunofluorescence of labeled taste cells was taken using FV1000 microscope and Fluoview software (Olympus).