Two- to 4-month-old C57Bl/6 mice of either sex were used in all experiments. Mice were maintained and sacrificed in accordance with protocols approved by the Animal Care and Use Committee at the University of Colorado, School of Medicine.
X-ray irradiation was delivered via an RS 2000 Biological Irradiator. Before irradiation, the mice were anesthetized with fresh Avertin i.p. (0.5 mg/gram mouse), and the body was shielded with lead, leaving only the head and neck exposed. In pilot experiments, we found that the typical 16 Gy dose used by others (Nelson, 1998
; Yamazaki et al., 2009
) resulted in 50% mortality by 1 dpi, and all animals were dead within 2 weeks (data not shown). We also tested lower doses of 1, 2 or 4 Gy, which were well tolerated by the mice, and produced similar, but smaller effects than the selected 8 Gy dose. Eight Gy was selected for further investigation as this dose was not lethal over the experimental period of 21 days, yet triggered a robust response in taste epithelium. Dose administration was calculated and adjusted by a dosimeter.
Mice were anesthetized as above, and perfused transcardially with 4% paraformaldehyde in 0.1M phosphate buffer (PFA). Tongues were dissected free from the lower jaw, and postfixed in 4% PFA overnight at 4°C, followed by immersion in sucrose (20% in 0.1M PB) overnight at 4°C. Cryoprotected tongues were embedded in OCT compound (Tissue Tek) and cryosectioned at 12 µm. Sections were thaw-mounted and stored at −20°C overnight before staining.
Immunofluorescence for taste cells
Sections were rehydrated in 0.1M PBS for 30 minutes, and blocked with blocking solution (0.2M PB, 0.05M NaCl, 0.1% triton X-100; 1% bovine serum albumin) with 5% normal goat serum for 2 hours at room temperature (except for goat anti-Car4 antiserum). Sections were then incubated overnight at 4°C in primary antiserum diluted in blocking solution without goat serum. Antisera include: (1) rabbit anti-Gustducin (1:1000; catalog #sc-395, Santa Cruz); (2) rabbit anti-Trpm5 (1:1000; gift from Dr. Emily Liman, USC); or (3) goat anti-Car4 (1:1000; catalog #AF2414, R&D Systems). Sections were then washed with PBS for 2 hours, and incubated in the appropriate secondary antiserum (goat anti-rabbit Alexa Fluor 546 (1:1000; Invitrogen), or donkey anti-goat Alexa Fluor 546 (1:1000; Invitrogen) in blocking solution for 2 hours at room temperature. Sections were washed in 0.1M PBS for 2 hours, counterstained with Sytox (Invitrogen), mounted in Fluoromount G and coverslipped for analysis using fluorescence and confocal microscopy.
Immunofluorescence for cell cycle markers
The method for phospho-histone3 (pH3) immunostaining has been described (Nguyen & Barlow 2010
). For Ki-67 immunofluorescence, sections were washed thrice in 0.1M PBS, treated with sodium citrate buffer (pH 6.0) at 95°C for 15 minutes, and cooled to room temperature for 30 minutes. Sections were then washed in PBS, incubated in blocking solution with 5% normal goat serum for 2 hours at room temperature, followed by 15 minutes each for avidin/biotin blocking solutions (A/B blocking kit; Vector Laboratories). Sections were then incubated in rabbit anti-Ki-67 antiserum (1:200; Thermo Scientific) overnight at 4°C. After 3 washes with PBS buffer for 1 hour each, sections were incubated with biotin-conjugated anti-rabbit IgG (Vector Laboratories) diluted 1:500 in PBS with 0.1% Tween 20 and 2.5% normal goat serum for 1 hour at room temperature. Sections were washed with PBS for 1 hour, then incubated in Streptavidin 546 (1:1000; Chemicon International) in PBS buffer for 2 hours at room temperature. After final washes in PBS for 1 hour, sections were counter-stained with Sytox green (Invitrogen) and mounted in Fluoromount G (Southern Biotech).
For BrdU immunofluorescence, sections were washed in 1T buffer (0.1M Tris pH 7.5, 0.15M NaCl), then incubated in a solution of 50% formamide and 5× SSC. After 3 washes in 1T buffer, sections were blocked in 1% blocking reagent in 1T buffer for 30 minutes at room temperature. Anti-BrdU monoclonal antibody (2μg/ml; catalog #10875400, Roche) was applied to tissues together with DNaseI (10UI/ml; Roche) for 50 minutes at 37°C. Sections were washed with 1T buffer, and incubated with goat-anti-mouse Alexa 546 for 1 hour at room temperature. After washing, sections were counter-stained with Sytox green and mounted.
TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) was performed using the In Situ Cell Death Detection kit TMR Red (Roche Applied Science). After 3 washes in PBS, sections were treated with 3% hydrogen peroxide in methanol, bathed in sodium citrate buffer (pH 6.0) at 95°C for 15 minutes, and then cooled to room temperature for 40 minutes. Sections were washed in PBS and permeabilized for 2 minutes on ice in a solution of 0.1% triton X-100 and 0.1% sodium citrate. Sections were incubated in blocking solution (20% normal goat serum, 3% bovine serum albumin, tris-HCl 50 mM at pH 7.5) for 30 minutes. After washing in PBS for 30 minutes, sections were incubated for 1 hour at 37°C with 10% enzyme solution diluted in buffer solution (Roche kit). Sections were washed in 0.1M PBS for 1 hour, counterstained with Sytox green (Invitrogen), mounted in Fluoromount G and coverslipped for analysis using fluorescence and confocal microscopy.
Birthdating of new taste cells
Mice were injected i.p. with 5-bromo-2-deoxyuridine (BrdU; Sigma; 120 mg/kg) twice, at 10 a.m. and at 1 p.m. For the 3 different series of birthdating experiments, BrdU injections were as follows: (i) the first BrdU dose was injected 6 hours before irradiation, and mice were then euthanized at 1 or 2 dpi (Figure 4); (ii) the first BrdU dose was injected at 5 dpi, and mice euthanized at 6 or 7 dpi (Figure 6A–E); or (iii) the first BrdU dose was injected at 6 dpi, and mice euthanized at 7 or 8 dpi (Figure 6F–J). Tongue cryosections (as above) from these mice were incubated in 90% methanol containing 3% hydrogen peroxide, washed in PBS, and then treated with 0.05% trypsin solution (37°C for 5 minutes). After washing in PBS, sections were treated with 4N HCl at 50°C for 15 minutes, then blocked with M.O.M. mouse Ig blocking reagent (Vector Laboratories; 1 hour at room temperature), and incubated overnight at 4°C with mouse anti-BrdU (1:500; catalog #G3G4, Developmental Studies Hybridoma Bank). After washing with PBS for 3 hours, sections were blocked in avidin/biotin blocking solutions (A/B blocking kit; Vector Laboratories), incubated for 60 minutes with biotin-conjugated anti-mouse IgG (1:500; Vector Laboratories), washed for 1 hour in PBS, and then incubated in ABC solution (Vector Laboratories) for 90 min at room temperature. Sections were washed for 1 hour, reacted with nickel-intensified DAB (Vector Laboratories) for 6–10 minutes, dehydrated with ethanol (50%, 70%, 95% and 100%), rinsed in xylene and coverslipped with Permount (Fisher Scientific). Newborn cells within taste buds were also identified using BrdU immunofluorescence and Sytox nuclear counterstain (see above).
Immunofluorescent images were obtained on an Olympus BX50 laser scanning confocal microscope. Images consisting of projected Z series of 0.75 μm optical sections were processed with Fluoview v5.0 software. Nomarski images were obtained with a Zeiss Axioplan 2 microscope equipped with an Axiocam cooled CCD camera and Axiovision imaging software.
The data for this study were gathered from 3 to 6 mice per time point. All quantitative measures of cells within taste buds were taken from taste bud profiles with taste pores or the middle profile of taste buds based on serial sections. For the entire study, all tallies and cell position assignments were done blind with respect to treatment and animal. Slides were identified only by number, and not the details of experimental or control treatment. The data were decoded and analyzed only after all tallies had been made.
The labeling index for each proliferation marker was calculated by dividing the number of immunopositive cells for each marker e.g. Ki67 immunoreactive (Ki67-IR), BrdU-IR, or pH3-IR, by the total number of basal epithelial cells (identified and quantified via Sytox labeling). Only basal keratinocytes along the basement membrane and within the trenches of circumvallate papillae that contain taste buds (e.g. Figure 1, basal keratinocytes lying between the arrowheads) were counted.
For BrdU birthdating studies, the border of taste buds was determined by (i) Nomarski imaging and (ii) fluorescence of Sytox green-stained nuclei. In order to include counts from individual taste bud profiles, each profile had to meet the following criteria: 1) the taste bud profile extended from the base of epithelium to its apex; and 2) if a taste bud extended across multiple sections, the middle, and therefore largest profile was selected, so that the counted profiles represent approximately the center of each taste bud. Only taste buds with clear borders were tallied in our analyses (Figures 4 and 6, taste buds with outlines). We then counted the number of BrdU-IR nuclei within each taste bud. Edge cells with elongate nuclei were considered to be outside of taste buds, whereas recently generated (24–48 hrs) BrdU+ taste bud cells were located inside the basal compartment of each bud (e.g. Figures 4A,B and 6A,C,F). BrdU+ cells outside of taste buds were also found in the basal and suprabasal compartments of non-taste epithelium within the circumvallate papilla (CVP) (examples in all panels of Figures 4 and 6).
In pilot studies comparing the average nuclear diameter of control basal epithelial cells with those from irradiated mice at 1, 3 and 5 dpi, we found that nuclear size varied significantly with radiation exposure (one-way ANOVA, n=3 mice per time point, p = 0.02). Thus to normalize the data across experimental conditions, we used Abercrombie correction (Abercrombie, 1946
) as follows: Z-stack projections with a total depth of 9.75 μm were obtained from 0.75 μm optical sections of 12 μm physical sections. The top and bottom optical sections were discarded to make sure tallied optical planes were physically complete, thus minimizing any problems due to variation in section thickness. The nuclear diameter parallel to the basal membrane, and thus perpendicular to the plane of section, of 20 randomly selected basal epithelial cells (enumerated and then selected via a random number generator) was measured and averaged. This average diameter for each condition was used in the Abercrombie formula using a section thickness of 9.75 μm. As cell cycle phase correlates with nuclear size, we reasoned that each marker class could comprise a subset of similarly sized nuclei whose average diameter would be distinct from that of the total basal population. In fact, we found that the average nuclear diameter for cells at different phases of the cell cycle, and thus the Abercrombie correction factor, was distinct from that of the Sytox population as a whole. Therefore, we elected to also correct counts for all markers at each time post irradiation.
A t-test or one-way ANOVA with a Tukey post hoc multiple comparisons test was used to analyze data after ascertaining the data were normally distributed using the Anderson-Darling test. Data are presented as mean+/− SD unless otherwise noted.