Of the 21 antibodies raised against mouse ORs, six yielded a consistent immunoreactive signal in the mouse MOE; of these six, two also stained specific glomeruli in the olfactory bulb..
Trang 15 Max Planck Research Unit for Neurogenetics,
6 Max-von-Laue-Strasse 4, D-60438 Frankfurt, Germany
7 Abstract—In the mouse, odorant receptor proteins (ORs) are
G-protein-coupled receptors expressed in mature olfactory
sensory neurons (OSNs) of the main olfactory epithelium
(MOE) ORs mediate odorant reception at the level of the
OSN cilia Most 1100 OR genes in the mouse genome are
expressed, at the RNA level, in mature OSNs The literature
on antibodies against ORs is limited, and most reports are
with antibodies that are not commercially available Here
we have screened 40 commercial antibodies against human
and mouse ORs by immunofluorescence staining of coronal
cryosections of the MOE of 21-day-old C57BL/6J mice.
Various methods of antigen retrieval were tested Of the 19
antibodies raised against human ORs, three yielded a
con-sistent immunoreactive signal in the mouse MOE; of these
three, two appeared to cross react against one or more,
unknown, mouse ORs Of the 21 antibodies raised against
mouse ORs, six yielded a consistent immunoreactive signal
in the mouse MOE; of these six, two also stained specific
glomeruli in the olfactory bulb Antibody specificity could
be validated with gene-targeted mouse strains in the case
of three ORs The number of OSNs immunoreactive for the
MOR28/Olfr1507 antibody is greater in C57BL/6J than in
129S6/SvEvTac wild-type mice Taken together, our results
are encouraging: 20–30% of these commercially available
antibodies are informative in immunohistochemical
analy-ses of the mouse MOE The commercial availability of these
antibodies should facilitate the study of OR proteins in the
MOE and the olfactory bulb, and the replicability of results
in the literature Ó 2016 The Author(s) Published by Elsevier
Ltd on behalf of IBRO This is an open access article under
the CC BY-NC-ND license ( http://creativecommons.org/
licenses/by-nc-nd/4.0/ ).
Key words: olfactory receptor, olfactory sensory neuron,
glomerulus, antibody, immunofluorescence, antigen retrieval.
8
9 INTRODUCTION
10
In mammals such as mouse and rat, odorants are
11
detected in the main olfactory epithelium (MOE) by
12
olfactory sensory neurons (OSNs) via odorant receptors
13
(ORs), which are G-protein-coupled
seven-14
transmembrane proteins (Buck and Axel, 1991) In the
15
mouse, there are 1099 OR genes with an intact open
16
reading frame, of which 1,087 are expressed at the
17
RNA level in OSNs (Saraiva et al., 2015)
18
Due to the difficulty in raising antibodies against
19
G-protein-coupled receptors, there have been relatively
20
few reports about OR proteins in the literature Initial
21
studies with antibodies against rat ORs suggested that
22
the antigen is localized to the cilia of OSNs in the rat
23
MOE (Koshimoto et al., 1992; Krieger et al., 1994) In situ
24
hybridization against OR RNAs in mouse revealed that
25
OR gene expression is restricted to OSNs within discrete
26
regions of the MOE called zones (Ressler et al., 1993;
27 Miyamichi et al., 2005) Gene-targeted mice enabled the
28
visualization of entire OSNs expressing a given OR by
29
targeted integration of a cassette that affords
cotransla-30
tion of the OR with a marker such as taulacZ via an
inter-31
nal ribosome entry site (IRES) (Mombaerts et al., 1996)
32
Axons of OSNs expressing the same OR coalesce into
33
two or a few glomeruli per mouse olfactory bulb, which
34
reside in highly restricted regions (Ressler et al., 1994;
35 Mombaerts et al., 1996; Zapiec and Mombaerts, 2015)
36
Replacement of the OR coding region showed that the
37
OR does not only mediate odorant detection (Bozza
38
et al., 2002), but is also a determinant of where in the
39
olfactory bulb the axons coalesce and form glomeruli
40
(Mombaerts et al., 1996; Feinstein et al., 2004; Feinstein
41
and Mombaerts, 2004)
42
The widespread success of the gene-targeted
43
approach of the OR-IRES-marker design may have
44
overshadowed or diminished efforts to raise and
45
characterize OR antibodies But these gene-targeted
46
mutations are designed to visualize the OSNs (including
47
their cilia, dendrite, cell body, axon, and axon terminals)
48
that express a given OR, and do not enable the
49
visualization of the subcellular localization of the OR
50
protein within the OSNs An attempt at creating an OR:
51
GFP C-terminal fusion protein by gene targeting
52
resulted, for reasons that remain unclear, in altered
53
positions of the corresponding glomeruli in the olfactory
54
bulb (Feinstein et al., 2004)
55
Antibodies against mouse ORs revealed that OR
56
proteins are present not only in OSN cilia, dendrite, and
57
cell body but also within the axon and axon terminals
http://dx.doi.org/10.1016/j.neuroscience.2016.12.044
*Corresponding author.
E-mail addresses: vicky.low@gen.mpg.de (V F Low),
peter.mom-baerts@gen.mpg.de (P Mombaerts).
Abbreviations: aa, amino acid; AR, antigen retrieval; EDTA,
ethylenediaminetetraacetic acid; GFP, green fluorescent protein;
IRES, internal ribosome entry site; MOE, main olfactory epithelium;
OMP, olfactory marker protein; OR, odorant receptor; OSN, olfactory
sensory neuron; PBS, phosphate-buffered saline; PFA,
paraformaldehyde; SDS, sodium dodecyl sulfate.
Neuroscience xxx (2017) xxx–xxx
1
Trang 258 (Barnea et al., 2004; Strotmann et al., 2004), thus
sup-59 porting the notion that the OR protein is involved in axonal
60 wiring of OSNs
61 A quarter of a century after the discovery of
62 mammalian OR genes (Buck and Axel, 1991), there are
63 still few papers on immunofluorescence staining of the
64 mouse MOE and olfactory bulb with OR antibodies that
65 are commercially available There are numerous
antibod-66 ies against ORs on the market, but they remain largely
67 uncharacterized The signal yielded by such antibodies
68 can be affected by the high sequence homology between
69 ORs, by post-translational modifications, by protein
fold-70 ing, by protein–protein interactions, and by fixation
71 crosslinking To increase the accessibility of the antigen
72 to the antibody, antigen retrieval can be used to break
73 some of the crosslinks formed during fixation and to alter
74 the protein re-folding following heat denaturation (Shi
75 et al., 2001; Emoto, 2005) Epitope unmasking through
76 antigen retrieval can allow for a more accurate detection,
77 and therefore representation, of the antigen
78 Here, we describe our screening of 40 commercially
79 available antibodies against human and mouse ORs in
80 the mouse MOE using various antigen retrieval
81 techniques, in coronal 12-lm sections of the MOE of
82 21- day-old C57BL/6J mice We obtained reliable
83 immunoreactive signals with two antibodies against
84 human ORs (due to presumed crossreactivity with
85 mouse ORs) and six antibodies against mouse ORs in
86 the mouse MOE Of these six, two antibodies also
87 stained specific glomeruli in the olfactory bulb
89 Mice
90 Mice were 21 days old Wild-type mice were C57BL/6J or
91 129S6/SvEvTac Our gene-targeted strains (Feinstein
92 et al., 2004) are publicly available from The Jackson
Labo-93 ratory (Bar Harbor, ME, USA): mI7-IRES-tauGFP (https://
94 www.jax.org/strain/006664), and
M50-IRES-GFP-IRES-95 taulacZ (https://www.jax.org/strain/006686) The
gene-96 targeted strain MOR28-IRES-gap-GFP (Serizawa et al.,
97 2000) is publicly available from the RIKEN BioResource
98 Center (Tsukuba, Japan) (
https://www2.brc.riken.jp/lab/an-99 imal/detail.php?brc_no=RBRC02928) Mice were
main-100 tained in specified pathogen-free conditions in individually
101 ventilated cages of the Tecniplast green line Mice received
102 ad libitum gamma-irradiated ssniff V1124-727 (ssniff,
103 Soest, Germany) Nesting, bedding, and enrichment were
104 provided as nestpak, Datesand Grade 6 (Datesand,
105 Manchester, United Kingdom) Mouse experiments were
106 performed in accordance with the German Animal Welfare
107 Act, European Communities Council Directive 2010/63/EU,
108 and institutional ethical and animal welfare guidelines of the
109 Max Planck Research Unit for Neurogenetics All efforts
110 were made to minimize the number of animals used and
111 their suffering
112 Immunohistochemical staining
113 Tissue preparation Mice were anesthetized by
114 injection of ketamine HCl and xylazine (120 mg/kg and
115
5 mg/kg body weight, respectively) and perfused with
116
5 ml ice-cold phosphate-buffered saline (PBS), followed
117
by 15 ml 2% paraformaldehyde in PBS (PFA) The
118
mouse heads were dissected, postfixed in 2% PFA for
119
2 h at 4°C, and decalcified in 0.45 M EDTA in PBS
120
overnight at 4°C Samples were cryoprotected in,
121
successively, 15% and 30% sucrose in PBS at 4°C,
122
frozen in O.C.T Compound (Tissue-Tek), sectioned at
123
12lm with a Leica CM3500 cryostat, and collected onto
124
glass slides Alternative fixation methods were also
125
tested; perfusion with 4% PFA resulted in poorer
126
antigenicity, and immersion fixation in 2% PFA for 2 h
127
without perfusion did not yield improved antigenicity
128
Antigen retrieval Various antigen retrieval steps were
129
tested to optimize visualization of the target protein
130
These included a 10-min incubation in 2 M HCl at room
131
temperature, or 10 min in ice-cold 100% methanol
132
followed by 0.5% sodium dodecyl sulfate (SDS) in PBS
133
at room temperature Antigen retrieval with a pressure
134
cooker (2100 Retriever; BioVendor) was carried out with
135
10 mM citric acid pH 6.0 and 10 mM Tris pH 9.0 with or
136
without 0.05% Tween 20 and 2 mM EDTA After heating
137
in a pressure cooker to 121°C, slides were left to cool
138
for 2 h in the retrieval buffer Additionally, PBS washes
139
were tested with 0.05% Tween 20 or 0.2% Triton X-100,
140
and incubation with antibody was tested for various
141
times during a period of 1–4 days, with and without
142
0.1% Triton X-100 Best results were obtained with PBS
143
washes and incubation with antibody diluted with 0.1%
144
Triton X-100 There was no obvious difference in
145
antigen detection with different incubation times
146
Immunohistochemistry Slides were washed
147
3 10 min with PBS, and, if necessary, antigen retrieval
148
was carried out and the slides were again washed
149
Slides were then blocked with 10% normal goat serum
150
(Jackson ImmunoResearch Laboratories) or normal
151
donkey serum (Millipore) in PBS for 1 h at room
152
temperature After blocking, slides were incubated with
153
the primary antibodies in 1% normal goat serum or
154
normal donkey serum with 0.1% Triton X-100 in PBS
155
overnight at 4°C Commercial rabbit antibodies against
156
ORs were tested at dilutions of 1:200, 1:500, and
157
1:2000 The following antibodies against human ORs
158
were obtained from Osenses (Keswick, Australia):
159
OR11H4, OR1B8, OR1D4, OR1E1, OR1L8, OR1M1,
160
OR1N2, OR2A4, OR2D3, OR2F1, OR2H1, OR2K2,
161
OR2T1, OR2V1, OR2W1, OR4C11, OR51E1, OR8A1
162
The following antibodies against mouse ORs were
163
obtained from Osenses: Olfr2, Olfr6 (x2; different
164
peptide sequences), Olfr15, Olfr16, Olfr24, Olfr56,
165
Olfr151, Olfr156, Olfr362, Olfr412, Olfr521, Olfr545,
166
Olfr552, Olfr554, Olfr685, Olfr749, Olfr831, Olfr1507
167
We also tested sheep OR51E1 and chicken Olfr73
168
antibodies (Osenses), and rabbit Olfr1303 antibody (Life
169
Technologies) Mature OSNs were visualized with goat
170
OMP antibodies (olfactory marker protein; 1:1000; Wako
171
Chemicals), and green fluorescent protein (GFP) was
172
detected with chicken GFP antibodies (1:1000; Aves
173
Labs) After incubation with primary antibodies, slides
Trang 3174 were washed with PBS, then incubated with secondary
175 antibodies: donkey goat Alexa488, donkey
anti-176 sheep Alexa488, goat anti-chicken Alexa488, donkey
177 anti-rabbit Alexa555, goat anti-rabbit Alexa594 (1:1000;
178 Invitrogen), and DAPI (1:10,000; Molecular Probes) for
179 1 h at room temperature Slides were washed with PBS
180 and coverslipped with Mowiol (Calbiochem)
181 Combined immunohistochemistry andin situ
182 hybridization
183 Tissue preparation Mice were anesthetized by
184 injection of ketamine HCl and xylazine (120 mg/kg and
185 5 mg/kg body weight, respectively) and perfused with
186 15 ml ice-cold 4% PFA The mouse heads were
187 dissected, postfixed in 4% PFA, and decalcified in
188 0.45 M EDTA in PBS overnight at 4°C Samples were
189 cryoprotected in 15% and 30% sucrose in PBS at 4°C,
190 frozen in O.C.T Compound (Tissue-Tek), sectioned at
191 12lm with a Leica CM3500 cryostat, and collected onto
192 glass slides
193 In situ hybridization and immunohistochemistry OR
194 RNAs were detected with biotinylated riboprobes
195 against Olfr156 (Strotmann et al., 1999); Olfr412 (forward
196 50-ATGGACGGAGGCAACCAGA-30, reverse 50-GGCTT
197 CCTTAGCAGTCTTCCC-30), and Olfr552 (Addgene
198 plasmid #15843; Hirota et al., 2007) Riboprobes were
199 generated as described inIshii et al (2004) Combined
200 immunohistochemistry and in situ hybridization was
car-201 ried out using a modified protocol from Ishii et al
202 (2004) Briefly, slides were washed with PBS and antigen
203 retrieval was carried out with 10 mM citric acid pH 6.0
204 Next the slides were fixed with 4% PFA for 15 min,
205 washed 5 min with PBS, followed by blocking
endoge-206 nous peroxidases with 0.1% H2O2 in PBS for 30 min
207 Slides were washed in PBS, incubated in 0.2 M HCl for
208 10 min, followed by another PBS wash Slides were then
209 incubated for 10 min in 0.1 M Triethanol amine-HCl,
210 0.25% acetic anhydrate; pH 8.0 After being washed in
211 PBS, slides were dehydrated in graduated ethanol and
212 left to air-dry Probes were diluted in hybridization solution
213 and the slides were incubated overnight at 65°C Slides
214 were then washed as described in Ishii et al (2004),
215
blocked with TNB (0.5% NEN Blocking reagent in TN;
216
100 mM Tris–Cl pH 7.5, 150 mM NaCl) for 1 h at room
217
temperature, and incubated with streptavidin–HRP
218
(1:1500; PerkinElmer) and an OR antibody: rabbit Olfr156
219
antibody, rabbit Olfr412 antibody, and rabbit Olfr552
anti-220
body (1:2000; Osenses) diluted in TNB with 0.1% Triton
221
X-100, for 3 nights at 4°C Slides were then washed
222
thoroughly with TN-T (TN with 0.05% Tween 20)
223
Streptavidin-labeled biotinylated probes were visualized
224
by incubation with tyramide-fluorescein (1:50;
PerkinEl-225
mer) diluted with 1 amplification diluent (PerkinElmer)
226
for 10 min Finally, in order to visualize the antibody,
227
slides were washed with TN-T and incubated with goat
228
anti-rabbit Alexa594 (1:1000; Invitrogen) and DAPI
229
(1:10,000) for 2 h at room temperature Slides were
230
washed with TN before coverslipping with Mowiol
231
(Calbiochem)
232
Image analysis
233
Microscopy Following labeling with
immunohisto-234
chemistry, and immunohistochemistry combined with
235
in situ hybridization, slides were examined under a
236
Zeiss LSM 710 confocal microscope
237
Cell counting Every fifth section from the mouse MOE
238
was labeled with rabbit Olfr1507 antibody (visualized with
239
Alexa 594) and chicken GFP antibody (visualized with
240
Alexa 488) Slides were scanned with a Pannoramic
241
Midi Scanner (3D Histech) The diameter of cell nuclei
242
was measured with Panoramic Viewer Software (3D
243
Histech) Each section was exported as a tiff file using
244
the Panoramic Viewer Software, and cells were counted
245
using the cell counter plugin in Fiji ImageJ version 1.50 g
246
Statistics Statistical analysis was performed using
247
GraphPad Prism 5 Values are shown as the mean ±
248
standard error of the mean A t-test was used to
249
compare between two means For multiple
250
comparisons, the one-way ANOVA Newman–Keuls
251
multiple comparison test was used to test for
252
significance Significance was set at P6 0.05
Table 1 List of antibodies against ORs
Antigen Host Antigen peptide region Catalogue Source Optimal antigen retrieval Optimal antibody dilution
OR1D4 Rabbit Not specified OSR00252W Osenses Citric acid AR 1:2000
Olfr6 Rabbit aa 275–316 OSO00046W Osenses Citric acid AR, Tris AR 1:2000
Olfr156 Rabbit Not specified OSR00044W Osenses Citric acid AR, methanol/SDS 1:2000
Olfr412 Rabbit aa 260–312 OSR00211W Osenses Citric acid AR 1:2000
Olfr552 Rabbit aa 270–317 OSR00204W Osenses No AR, Citric acid AR 1:2000
Olfr1507 Rabbit aa 265–313 OSR00212W Osenses No AR, Citric acid AR 1:5000
List of commercial antibodies raised against human and mouse ORs that yielded immunostaining in cryosections of the mouse MOE As the peptide sequence of the antigens
is not available, the region is given from which the antigenic peptide was designed Antigen retrieval conditions, and optimal dilutions of the antibodies are indicated.
aa; amino acid, Citric acid AR; Antigen retrieval with citric acid buffer pH 6.0 in a pressure cooker, No AR; No antigen retrieval, Tris AR; Antigen retrieval with Tris buffer pH 9.0 in a pressure cooker.
Trang 4253 RESULTS
254 We screened 40 commercially available antibodies that
255 were raised against human or mouse ORs in the mouse
256 MOE, testing various antigen retrieval methods to
257 optimize visualization of the antigen Of these 40
258 antibodies, 19 were raised against human ORs with
259 80% amino acid identity to mouse ORs, and 21 were
260 against mouse ORs Here we present the results from 9
261 of the antibodies that yielded reproducible
immuno-262 staining in the mouse MOE (Table 1) For the sake of
263 brevity, we refer to the antibody with the name of the OR
264 against which it was raised: ORxxx for human ORs, and
265 Olfrxxx for mouse ORs
266 Screening of antibodies against
267 human ORs in the mouse MOE
268 Of the 19 antibodies against human
269 ORs, three yielded consistent
270 immunoreactivity when antigen
271 retrieval was carried out in a
272 pressure cooker with citric acid:
273 OR1D4, OR1L8, and OR51E1 Both
274 the OR1D4 antibody (Fig 1A, B0) and
275 the OR1L8 antibody (Fig 1C, D0)
co-276 labeled with the OMP antibody,
277 suggesting that they cross react with
278 one or more mouse OR proteins in
279 the MOE The mouse ORs with the
280 closest homology to these human
281 ORs are Olfr412 (81% identity with
282 OR1D4 across the entire amino acid
283 sequence of the OR) and Olfr355
284 (78% identity with OR1L8 in the
285 amino acid region 200–250 from
286 which the antigenic peptide was
287 designed) For the OR1D4 and
288 OR1L8 antibodies, immunoreactivity
289 was observed midway along the
290 dorsal-medial and ventral-lateral
291 aspect of the MOE OR51E1
292 immunoreactivity was observed more
293 ventral-laterally The rabbit OR51E1
294 antibody reacted with an antigen in
295 the mouse MOE (OR51E1 has 87%
296 identity with mouse Olfr558 in the
297 amino acid region 270–317 from
298 which the antigenic peptide was
299 designed): signal was detected in cell
300 bodies, but there was no evidence of
301 immunoreactivity in cilia or dendrites
302 Furthermore OR51E1
immuno-303 reactivity did not colocalize with OMP
304 immunoreactivity (Fig 1E, F0)
305 Screening of antibodies against
306 mouse ORs in the mouse MOE
307 Of the 21 antibodies against mouse
308 ORs, we observed consistent
309 immunoreactivity with six antibodies:
310
Olfr2, one of the Olfr6 antibodies (OSO00046W),
311
Olfr156, Olfr412, Olfr552, and Olfr1507
312
The Olfr2 and Olfr6 antibody labeling was observed in
313
the ventral-lateral aspect of the MOE, and appeared weak
314
(Fig 2) Olfr2 labeling was best observed after antigen
315
retrieval in the pressure cooker with citric acid, and
316
Olfr6 labeling was observed with both citric acid and
317
Tris antigen retrieval buffers in the pressure cooker Low
318
signal could be due to low protein expression of the
319
Olfr2 and Olfr6 ORs, low sensitivity of the antibodies, or
320
suboptimal fixation and antigen retrieval procedures
321
Fortunately, there are gene-targeted mouse strains
322
available for these two ORs, respectively
mI7-IRES-323
tauGFP (Bozza et al., 2002) and
M50-IRES-GFP-IRES-Fig 1 Labeling of the mouse MOE by antibodies against human ORs (A) OR1D4-immunoreactive cells in the MOE (red) Mature OSNs were detected with OMP antibody (green) Merge with DAPI (blue) (B) High magnification of insert in A showing weak OR1D4 antibody labeling in the dendrite and cell body of OMP-immunoreactive cells in the MOE (merged red and green, B0) (C) OR1L8-immunoreactive cells labeled in the MOE (red) Mature OSNs were detected with OMP antibody (green) Merge with DAPI (blue) (D) High magnification of insert in C Antibody labeling is observed
in the cilia and cell body, and colocalized with OMP-immunoreactive cells (merged red and green,
D0) (E) OR51E1 labeling was detected in a population of cells in the MOE (red) Mature OSNs were detected with OMP antibody (green) Merge with DAPI (blue).(F) High magnification of insert in E demonstrating strong OR51E1 immunoreactivity in the cell bodies of a population of cells that were not OMP-immunoreactive (merged red and green, F0) (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.)
Trang 5324 taulacZ (Feinstein et al., 2004) These strains enabled us
325 to validate that Olfr2 (Fig 2A, B0) and Olfr6 (Fig 2D, E0)
326 antibody labeling was limited to cells that were also
327 GFP positive, indicating that the antibody is specific to
328 the respective OR Neither antibody against Olfr2
329 (Fig 2C, C0) nor Olfr6 (Fig 2D, D0) detected protein
330 expression in the GFP-positive glomeruli in the olfactory
331 bulb
332 The Olfr156 antibody yielded signal midway along the
333 dorsal-medial and ventral-lateral aspect of the MOE, and
334 appeared to have low immunoreactivity (Fig 3A, B0)
335 SDS/methanol antigen retrieval sporadically reveled
336 positive labeling, and citric acid antigen retrieval yielded
337 more reproducible signal In both cases only cilia were
338 labeled consistently, and the cell body, and dots within
339 the dendrites, were visible on rare occasions It is
340 possible that the antigen is more accessible in certain
341 subcellular localizations; there may be modifications
342 occurring to the OR protein that allow detection under
343 certain conditions; or an increased density of OR protein
344 in certain subcellular localizations, such as in the cilia,
345 may make it more easy to visualize
346 The Olfr412 antibody showed strong immunoreactivity
347 following citric acid antigen retrieval, and signal was
348 detected in the dorsal-medial aspect of the mouse
349 MOE (Fig 3C, D0) This pattern differs from the
350 OR1D4 antibody, which yielded signal midway along the
351
dorsal-medial and ventral-lateral aspect of the MOE
352
Thus, although Olfr412 is the mouse OR with the
353
highest homology (80%) to human OR1D4, it is unlikely
354
to be the antigen that the OR1D4 antibody detects
355
The Olfr552 antibody also showed strong
356
immunoreactivity in the mouse MOE (Fig 3E, F0) This
357
antibody did not require antigen retrieval, but the signal
358
was greatly improved following antigen retrieval with
359
citric acid Signal was detected through the
dorsal-360
medial aspect of the mouse MOE Two glomeruli were
361
detected in the olfactory bulb Consistent with Olfr552
362
belonging to class I ORs, the labeled glomeruli resided
363
within the class I dorsal domain of the olfactory bulb
364
The medial glomerulus was located midway along the
365
anterior-posterior extent, and the lateral glomerulus
366
(Fig 3G) was located more anteriorly
367
For Olfr156, Olfr412, and Olfr552, there are no
gene-368
targeted mice available to validate that the antibody is
369
specific for the respective OR Instead we used in situ
370
hybridization combined with immunohistochemistry to
371
verify that these antibodies were labeling the intended
372
OR Combining these methods compromises the quality
373
of both techniques, and we were unable to detect
374
antibody labeling with Olfr156 in combination with in situ
375
hybridization We were able to detect both protein and
376
RNA in the mouse MOE for Olfr412 (Fig 4A, B0) and
377
Olfr552 (Fig 4C, D0) We could thus verify that Olfr412
Fig 2 Labeling of the mouse MOE by antibodies against mouse ORs Olfr2 and Olfr6 (A) Olfr2-immunoreactive cells (red) in the MOE of an mI7-IRES-tauGFP / mouse These cells were also detected with GFP antibody (green) Merge with DAPI (blue) (B) High magnification of insert in A showing Olfr2 immunoreactivity in the cilia, dendrite, and cell body, and colocalization with GFP antibody signal (merged red and green, B0) (C) Olfr2 immunoreactivity was not detected in a GFP-immunoreactive (green) glomerulus of an mI7-IRES-tauGFP / mouse Merge with DAPI (blue) (C0) (D) Olfr6 immunoreactivity (red) was weak, and only occasionally detected in association with GFP immunoreactivity (green) in an M50-IRES-GFP-IRES-taulacZ / mouse Merge with DAPI (blue) (E) High magnification of insert in D Weak Olfr6 immunoreactivity is observed in the cell body, dendrite and cilia of cells that express M50-IRES-GFP-IRES-taulacZ (merged red and green, E0) (F) Olfr6 immunoreactivity was not detected
in GFP-immunoreactive (green) glomeruli of an M50-IRES-GFP-IRES-taulacZ / mouse Merge with DAPI (F 0 ) Scale bar = 20 lm (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Trang 6378 and Olfr552 antibody labeling was closely associated with
379 the signal from respective riboprobes in the mouse MOE,
380 indicating the antibodies are labeling the cells that
381 express the respective OR gene The sensitivity and
382 specificity of these antibodies could not be verified using
383 this method of combined labeling
384 Finally, the Olfr1507 antibody showed strong
385 immunoreactivity in the ventral-lateral aspect of the
386 mouse MOE (Fig 5), and did not require antigen
387 retrieval to be visualized Signal could be detected in the
388 cilia, dendrite, and cell body of OSNs Immunostaining
389 colocalized with GFP expression in mice homozygous
390 for the MOR28-IRES-gap-GFP gene-targeted mutation
391 (Serizawa et al., 2000) (Fig 5A, B0) We also detected
392
strong immunoreactivity in
GFP-393
positive glomeruli in the olfactory bulb
394
(Fig 5C, C0) Having a reliable
anti-395
body that detects a mouse OR for
396
which we have a gene-targeted
397
mouse strain available gave us the
398
opportunity to investigate monoallelic
399
expression of Olfr1507 in mice
400
heterozygous for
MOR28-IRES-gap-401
GFP, with the other, wild-type
402
MOR28/Olfr1507 allele either of
403
C57BL/6J origin (abbreviated B6) or
404
129S6/SvEvTac origin (abbreviated
405
129) (Fig 5D, E0) The MOR28/
406
Olfr1507 gene targeting had been
407
carried out in an embryonic stem cell
408
line of 129 origin (Serizawa et al.,
409
2000) We observed an even
distribu-410
tion of cells that are doubly GFP
411
positive and antibody positive
412
throughout the MOE, versus cells that
413
are antibody-only positive In the
414
olfactory bulb we found that the
415
GFP-positive glomeruli were labeled
416
with the Olfr1507 antibody in
417
MOR28-IRES-gap-GFP+/ mice
418
(Fig 5F, F0) The glomeruli did not
419
appear to be homogeneously labeled
420
with Olfr1507 and GFP antibodies in
421
MOR28-IRES-gap-GFP+/ mice
422
Instead, there is a segregation of
423
axon terminals expressing the
gene-424
targeted and wild-type Olfr1507
alle-425
les This segregation was observed
426
in MOR28-IRES-gap-GFP+/ (129)
427
mice (Fig 5F’) and in
MOR28-IRES-428
gap-GFP+/ (B6) mice (data not
429
shown)
430
Counting
Olfr1507-immuno-431
reactive cells in the MOE
432
We counted
Olfr1507-immuno-433
reactive cells in every fifth coronal
434
section of the MOE from five types
435
of mice, 20 in total: MOR28-IRES-g
436
ap-GFP/ (n = 5),
MOR28-IRES-437
gap-GFP+/ (B6) (n = 4),
MOR28-438
IRES-gap-GFP+/ (129) (n = 3),
439
wild-type C57BL/6J (n = 5), and wild-type 129S6/
440
SvEvTac (n = 3) mice To correct for overcounting in
441
sections, we performed an Abercrombie correction
442
(Abercrombie, 1946; Bressel et al., 2016) (Table 2) We
443
observed no significant difference in the nuclear diameter
444
between strains, and used a correction factor of
445
0.68–0.69
446
In all five types of mice, we observed a distinct
447
‘M’-pattern distribution of Olfr1507-immunoreactive
448
OSNs in the MOE, with the peaks corresponding to the
449
ectoturbinates of the MOE (Fig 6A) The first
Olfr1507-450
immunoreactive cells were observed midway along the
451
anterior–posterior dimension, between 3.5 and 4 mm
Fig 3 Labeling of the mouse MOE by antibodies against mouse ORs Olfr156, Olfr412, and
Olfr552 (A) Olfr156-immunoreactive cells (red) Mature OSNs were detected with the OMP
antibody (green) Merge with DAPI (blue) (B) High magnification of insert in A showing Olfr156
immunoreactivity in the cilia and dendrite Merge with OMP immunoreactivity (B0) (C)
Olfr412-immunoreactive cells (red) Mature OSNs were detected with OMP (green) Merge with DAPI
(blue) (D) High magnification of insert in C showing Olfr412 immunoreactivity in the cilia, dendrite
and cell body, colocalizing with OMP immunoreactivity (merged, D0) (E) Olfr552-immunoreactive
cells (red) Mature OSNs were detected with OMP (green), and merged with DAPI (blue) (F) High
magnification of insert in E showing strong Olfr552 immunoreactivity in the cilia, dendrite and cell
body of cells that are labeled with OMP antibody (F0) (G) Olfr552 immunoreactivity was also
detected in a glomerulus Merge with DAPI Scale bar = 20 lm (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
Trang 7452 from the start of the MOE The expression peaked after
453 approximately 1 mm, then began to drop off, but
454 expression quickly increased again, and continued to
455 the most posterior portion of the MOE
456 In MOR28-IRES-gap-GFP/ mice, we found that
457 0.94 ± 0.4% of the counted cells were labeled with only
458 the Olfr1507 antibody but not with the GFP antibody,
459 suggesting that the antibody is highly specific for
460 Olfr1507-expressing cells in the mouse MOE
461 Conversely, in these mice only 0.40 ± 0.2% of the
462 counted cells were labeled with the GFP antibody but
463 not with the Olfr1507 antibody, indicating that the
464 antibody is also highly sensitive Therefore, we conclude
465 that the antibody against an Olfr1507 peptide that is
466 commercially available from Osenses (OSR00212W) is
467 a reliable, specific, and sensitive detector of
Olfr1507-468 expressing OSNs in the mouse MOE, with 98.66 ±
469 0.4% of counted cells labeled with both the Olfr1507
470 antibody and the GFP antibody in MOR28-IRES-gap-GF
471 P/ mice (Fig 6B)
472 Next, we examined the two populations of
Olfr1507-473 expressing OSNs for each allele in
MOR28-IRES-gap-474 GFP+/ mice OSNs that were labeled with both the
475 Olfr1507 and GFP antibodies were considered to
476 express the MOR28-IRES-gap-GFP gene-targeted
477 allele, and OSNs that were labeled with only the
478 Olfr1507 antibody were considered to express the
479 wild-type allele In both MOR28-IRES-gap-GFP+/
480
(B6) and MOR28-IRES-gap-GFP
481
+/ (129) mice, we observed a
482
homogeneous distribution of the two
483
populations throughout the MOE
484
(Fig 6A) However, there was a
485
slight preference for expression of
486
the wild-type, B6-derived allele in
487
MOR28-IRES-gap-GFP+/ (B6)
488
mice, with 56.38 ± 1.9% of cells
489
counted expressing the wild-type
490
Olfr1507 allele and 43.01 ± 1.8% of
491
cells expressing the mutant
492
Olfr1507 allele (P < 0.002; Fig 6B)
493
(An additional 0.62 ± 0.4% of the
494
cells counted reacted with the GFP
495
antibody only.) In
MOR28-IRES-496
gap-GFP (129) mice, we found that
497
51.12 ± 0.8% of cells expressed
498
the wild-type, 129-derived allele and
499
47.21 ± 0.1% of cells expressed
500
the gene-targeted allele
501
(P < 0.008) (An additional 1.67 ±
502
0.8% of cells counted reacted with
503
the GFP antibody only.) Although
504
the expression of the 129 wild-type
505
vs gene-targeted allele is still
506
statistically different, it is closer to
507
the expected 50/50 distribution than
508
when the wild-type allele is of B6
509
origin Note that expression of the
510
gap-GFP reporter is driven by a
511
129-derived promoter sequence in
512
the gene-targeted mutation
513
(Serizawa et al., 2000)
514
Further support for the idea that
515
the probability of expression of Olfr1507 is higher for the
516
B6 allele than for the 129 allele, came from comparing
517
the numbers of immunoreactive cells in the various
518
strains In MOR28-IRES-gap-GFP/ mice we counted
519
31,657 ± 2305 Olfr1507-immunoreactive cells, and
520
these numbers were not statistically different from
521
MOR28-IRES-gap-GFP+/ (129) mice (36,897 ±
522
1547), or 129S6/SvEvTac wild-type mice (24,985 ±
523
1448) (Fig 6C) Likewise, the numbers of cells counted
524
in MOR28-IRES-gap-GFP+/ (B6) mice (41,988 ±
525
3142) and in C57BL/6 wild-type mice (43,500 ± 3606)
526
were not significantly different from each other, but were
527
significantly higher than in MOR28-IRES-gap-GFP/
528
mice and in 129S6/SvEvTac wild-type mice (one-way
529
ANOVA P < 0.003; Fig 6C) Incidentally, we did not
530
identify a significant correlation between the number of
531
Olfr1507-immunoreactive OSNs counted in the MOE
532
and the weight of the mice, indicating that the
533
differences in cell numbers were not due to trivial
534
variations in mouse weight (Fig 6D)
535 DISCUSSION
536
Here, we have demonstrated that some commercially
537
available antibodies against ORs can be a reliable tool
538
to examine cells expressing a given OR in the mouse
539
MOE
Fig 4 In situ hybridization combined with immunohistochemistry in the mouse MOE (A) The
same sells were detected with the Olfr412 antibody (red) and the Olfr412 riboprobe (green) Merge
with DAPI (blue) (B) High magnification of insert in A showing Olfr412 immunoreactivity in the cilia,
dendrite, and cell body, and colocalizing with Olfr412 riboprobe hybridization in the cell body (B0).
(C) Cells were detected with Olfr552 antibody (red) and Olfr552 riboprobe (green) Merge with DAPI
(blue) (D) High magnification of insert in C showing Olfr552 immunoreactivity in the cilia, dendrite,
and cell body, and colocalizing with Olfr552 riboprobe hybridization detected in the cell body (D0).
Scale bar = 20 lm (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
Trang 8540 Antibodies against ORs
541 We have demonstrated that a substantial fraction (at least
542 20%) of commercially available antibodies are a viable
543 way for assessing OR expression in the mouse MOE
544 We found that tissue fixation had an effect on the quality
545 of antigen detection, with perfusion with 2% PFA being
546 optimal For the antibodies that we were unable to get
547 to work, other fixation methods may yield better and
548 consistent signals We also tested various antigen
549 retrieval methods, and found that when antigen retrieval
550
was required, the best results were often obtained with
551
antigen retrieval in a pressure cooker with a citric acid
552
buffer, pH 6.0 While alternative antigen retrieval
553
methods also worked, we found that if signal could still
554
not be detected after antigen retrieval with citric acid,
555
alternative methods would also be without success
556
Some antibodies yielded strong signals, and for others
557
the signal was barely or inconsistently detectable It
558
would be interesting to determine if the variation in
559
signal is a result of variable antigenicity of the
560
antibodies, or reflects widely differing levels of protein
561
expression within OSNs expressing these particular ORs
562
We confirm and extend the observations of OR
563
immunoreactivity in the cell body and dendrite as well
564
as in the cilia of OSNs (Barnea et al., 2004; Strotmann
565
et al., 2004) In the dendrite, and sometimes in the cell
566
body of OSNs, a distinct dot-like pattern could be
567
detected This pattern of signal could be an artifact of
anti-568
body staining Nevertheless, it is a repeatable observation
569
with various antibodies against ORs, and we reported it
570
earlier in the M71::GFP fusion mouse strain (Feinstein
571
et al., 2004), suggesting that this dot-like pattern of OR
572
protein signal reflects a biological process It could be
573
related to the visualization of specific forms of protein
574
folding, it may be due to increased density of protein as
575
a result of pulsed transcription, or it may be related to
pro-576
tein trafficking This issue can be examined further
Fig 5 Labeling of the MOE of MOR28-IRES-gap-GFP / and +/ mice by antibodies against Olfr1507/MOR28 (A) Olfr1507-immunoreactive cells (red) in a MOR28-IRES-gap-GFP / mouse are colabeled with GFP antibody (green) Merge with DAPI (blue) (B) High magnification of insert in A Olfr1507 immunoreactivity is detected in the cell body, dendrite, and cilia, and colocalizes with GFP immunoreactivity (B0) (C) Olfr1507 immunoreactivity colocalizes uniformly with GFP immunoreactivity within a glomerulus of a MOR28-IRES-gap-GFP / mouse Merge with DAPI (C’) (D) Olfr1507-immunoreactive cells (red) in a MOR28-IRES-gap-GFP+/ (129) mouse are colabeled with GFP antibody (green) Merged with DAPI (blue) (E) High magnification of insert in D Olfr1507 immunoreactivity is detected in the cell body, dendrite, and cilia, and colocalizes with GFP immunoreactivity (E’) (F) Olfr1507 immunoreactivity (red) segregates within a GFP-immunoreactive (green) glomerulus of an MOR28-IRES-gap-GFP+/ (129) mouse Merge with DAPI (F 0 ) Scale bar = 20 lm (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)
Table 2 Abercrombie correction; Nucleus diameter of OSNs
diameter ( lm)
Abercrombie correction factor MOR28-IRES-gap-GFP / 5.60 ± 0.13 0.68
MOR28-IRES-gap-GFP+/ (B6) 5.46 ± 0.11 0.69
MOR28-IRES-gap-GFP+/
(129)
5.43 ± 0.10 0.69
129S6/SvEvTac 5.30 ± 0.09 0.69
The average nucleus diameter and the standard error of the mean and the
calculated Abercrombie factors are given for the five types of mice Scale
bars = 20 lm.
Trang 9577 through the use of antibodies against ORs in combination
578 with membrane markers, and with super-resolution
579 microscopy
580 In situ hybridization combined with
581 immunohistochemistry
582 Due to the high homology of mouse ORs, a gene-targeted
583 mouse strain is ideal to validate specificity and to assess
584 sensitivity of an OR antibody We have demonstrated that
585 in some cases immunohistochemistry can be combined
586 with in situ hybridization as a form of validation, but this
587 technique has its limitations It can indicate that an
588 antibody is targeting the population of ORs that is
589 intended, but until this method is further improved, it
590 cannot reveal how specific or sensitive an antibody is
591 Correlating OR RNA expression with the corresponding
592 OR protein expression could reveal interesting information
593 about OR transcription and translation within OSNs
594 Glomerular labeling
595 In most cases we were unable to detect a reliable signal in
596 glomeruli But with 3,600 glomeruli in the mouse olfactory
597 bulb (Richard et al., 2010), it remains possible that we
598 overlooked the glomerular signal for some antibodies
599 For two antibodies (Olfr552 and Olfr1507/MOR28) we
600 were able to visualize specific glomeruli Interestingly,
601 both of these antibodies did not require antigen retrieval
602 to be detected Even when antigen retrieval was
603 performed, the glomeruli were still detected, suggesting
604 that antigen retrieval itself was not inhibiting the
605 detection of glomeruli with the other antibodies
606
In both MOR28-IRES-gap-GFP+/ (B6) and
607
MOR28-IRES-gap-GFP+/ (129) mice, we observed a
608
compartmentalization of immunoreactive signal within
609
the glomeruli: they were labeled uniformly with the
610
Olfr1507 antibody, but visualized only partially with the
611
GFP antibody The segregation in glomeruli in
MOR28-612
IRES-gap-GFP+/ (B6) mice has been reported
613
previously (Ishii et al., 2001), and there is a single amino
614
acid polymorphism between B6 (isoleucine) and 129
615
(valine) at residue 50 Interestingly, we also observed a
616
compartmentalization of the two populations of OSNs in
617
glomeruli of MOR28-IRES-gap-GFP+/ (129) mice This
618
compartmentalization may reflect a subtle difference in
619
RNA or protein expression level between the wild-type
620
and 129 alleles, or an undocumented effect of the
gap-621
GFP axonal marker In this marker, the 20 N-terminal
622
amino acid residues of GAP43 are fused to the
623
N-terminus of the GFP to target it to the plasma
mem-624
brane (Moriyoshi et al., 1996)
625
Probability of OR gene expression
626
We used MOR28-IRES-gap-GFP/ mice (Serizawa
627
et al., 2000) as validation that the Olfr1507 antibody is
628
highly specific and sensitive for Olfr1507-expressing
629
OSNs We also determined that Olfr1507-expressing
630
OSNs are distributed in an ‘M’-like pattern in the MOE in
631
a series of coronal sections We have described a similar
632
pattern of distribution of OSNs in the MOE for OSNs
633
expressing mI7/Olfr2, M50/Olfr6, or SR1/Olfr124
634
(Bressel et al., 2016) The number of
Olfr1507-635
expressing OSNs in a 21-day-old C57BL/6J mouse
Fig 6 Numbers of Olfr1507-immunoreactive cells in the MOE of wild-type and gene-targeted mice (A) Average number of cells counted per coronal section through the anterior–posterior dimension of the MOE in five types of mice: MOR28-IRES-gap-GFP /, MOR28-IRES-gap-GFP+/
(B6), MOR28-IRES-gap-GFP+/ (129), C57BL/6J, and 129S6/SvEvTac Cells labeled with the Olfr1507 antibody (red), the GFP antibody (green), and both the Olfr1507 and GFP antibodies (orange) (B) Percentages of cells labeled with the Olfr1507 antibody (red), the GFP antibody (green), and both the Olfr1507 and GFP antibodies (orange) in three types of mice: MOR28-IRES-gap-GFP /, MOR28-IRES-gap-GFP+/ (B6), and MOR28-IRES-gap-GFP+/ (129) mice (C) Comparison of the number of cells counted in each strain Cell counts in every fifth section were multiplied by five, and subjected to Abercrombie correction (D) There is no correlation between the numbers of Olfr1507-immunoreactive cells and mouse weight (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Trang 10636 (43,500 ± 3606) is slightly higher than the number of
637 MOR256-17-expressing OSNs (37,023 ± 6318)
638 (Bressel et al., 2016)
639 When we assessed Olfr1507 expression in
MOR28-640 IRES-gap-GFP+/ (B6) mice, we found that there was
641 a preferential expression of the wild-type, B6-derived
642 allele We observed a reduced preference of expression
643 of the wild-type, 129-derived allele in
MOR28-IRES-gap-644 GFP+/ (129) mice When we compared the number
645 of Olfr1507-expressing OSNs in wild-type mice, we
646 found that there were more cells in B6 mice than in 129
647 mice, suggesting that the probability of Olfr1507 gene
648 choice is higher when driven by the B6 promoter than
649 the 129 promoter There does appear to be an
650 additional effect of the targeted mutations in terms of a
651 slight reduction in the probability of gene choice
652 Interestingly, Olfr1507 is the most proximal OR gene
653 among a cluster of seven OR genes that are regulated
654 by the H element (Tsuboi et al., 1999; Serizawa et al.,
655 2000, 2003) When the H element is deleted by gene
tar-656 geting, the expression of Olfr1507 is abolished entirely,
657 and the expression of the more distal OR genes is
658 reduced in a distance-correlated fashion (Fuss et al.,
659 2007; Nishizumi et al., 2007; Khan et al., 2011)
More-660 over, the position of the Olfr1507 coding region in
661 C57BL/6 mice is 11 kb closer to the H element compared
662 to mice with a 129 background (Fuss et al., 2007) We
663 speculate that the difference in distance of the H element
664 to the Olfr1507 gene is causally related to the difference in
665 probability of gene choice, measured operationally as the
666 number of Olfr1507-expressing OSNs
668 Antibodies against mouse ORs are a valuable tool for
669 understanding the biological distribution of OR proteins,
670 and to characterize OR-specific glomeruli in the olfactory
671 bulb when no gene-targeted strain is available Their value
672 to olfactory neuroscience may have been overshadowed
673 by the success of the gene-targeted approach The
674 significance of reports based on custom-designed or
675 home-made polyclonal antibodies is constrained by the
676 lack of the commercial or public availability of these
677 reagents, hampering replicability studies Here we have
678 characterized 40 commercially available antibodies
679 against ORs We have demonstrated that they have a
680 reasonable probability of success for immunofluorescence
681 in the MOE (20–30%), and can be highly specific and
682 sensitive The production of monoclonal antibodies against
683 ORs will be an important next step
684 Acknowledgment—P.M acknowledges generous support from
685 the Max Planck Society.
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