NSC 17525 No. of Pages 11 7 January 2017 Please cite this article in press as: Low VF, Mombaerts P. Odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb. Neu- roscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2016.12.044 1 Neuroscience xxx (2017) xxx–xxx 2 ODORANT RECEPTOR PROTEINS IN THE MOUSE MAIN OLFACTORY 3 EPITHELIUM AND OLFACTORY BULB 4 VICTORIA F. LOW AND PETER MOMBAERTS * INTRODUCTION 9 5 Max Planck Research Unit for Neurogenetics, In mammals such as mouse and rat, odorants are 10 6 Max-von-Laue-Strasse 4, D-60438 Frankfurt, Germany detected in the main olfactory epithelium (MOE) by 11 olfactory sensory neurons (OSNs) via odorant receptors 12 7 Abstract—In the mouse, odorant receptor proteins (ORs) are (ORs), which are G-protein-coupled seven- 13 G-protein-coupled receptors expressed in mature olfactory transmembrane proteins (Buck and Axel, 1991). In the 14 sensory neurons (OSNs) of the main olfactory epithelium mouse, there are 1099 OR genes with an intact open 15 (MOE). ORs mediate odorant reception at the level of the reading frame, of which 1,087 are expressed at the 16 OSN cilia. Most 1100 OR genes in the mouse genome are RNA level in OSNs (Saraiva et al., 2015). 17 expressed, at the RNA level, in mature OSNs. The literature Due to the difficulty in raising antibodies against 18 on antibodies against ORs is limited, and most reports are with antibodies that are not commercially available. Here G-protein-coupled receptors, there have been relatively 19 we have screened 40 commercial antibodies against human few reports about OR proteins in the literature. Initial 20 and mouse ORs by immunofluorescence staining of coronal studies with antibodies against rat ORs suggested that 21 cryosections of the MOE of 21-day-old C57BL/6J mice. the antigen is localized to the cilia of OSNs in the rat 22 Various methods of antigen retrieval were tested. Of the 19 MOE (Koshimoto et al., 1992; Krieger et al., 1994). In situ 23 antibodies raised against human ORs, three yielded a con- hybridization against OR RNAs in mouse revealed that 24 sistent immunoreactive signal in the mouse MOE; of these OR gene expression is restricted to OSNs within discrete 25 three, two appeared to cross react against one or more, regions of the MOE called zones (Ressler et al., 1993; 26 unknown, mouse ORs. Of the 21 antibodies raised against Miyamichi et al., 2005). Gene-targeted mice enabled the 27 mouse ORs, six yielded a consistent immunoreactive signal visualization of entire OSNs expressing a given OR by 28 in the mouse MOE; of these six, two also stained specific glomeruli in the olfactory bulb. Antibody specificity could targeted integration of a cassette that affords cotransla- 29 be validated with gene-targeted mouse strains in the case tion of the OR with a marker such as taulacZ via an inter- 30 of three ORs. The number of OSNs immunoreactive for the nal ribosome entry site (IRES) (Mombaerts et al., 1996). 31 MOR28/Olfr1507 antibody is greater in C57BL/6J than in Axons of OSNs expressing the same OR coalesce into 32 129S6/SvEvTac wild-type mice. Taken together, our results two or a few glomeruli per mouse olfactory bulb, which 33 are encouraging: 20–30% of these commercially available reside in highly restricted regions (Ressler et al., 1994; 34 antibodies are informative in immunohistochemical analy- Mombaerts et al., 1996; Zapiec and Mombaerts, 2015). 35 ses of the mouse MOE. The commercial availability of these Replacement of the OR coding region showed that the 36 antibodies should facilitate the study of OR proteins in the OR does not only mediate odorant detection (Bozza 37 MOE and the olfactory bulb, and the replicability of results et al., 2002), but is also a determinant of where in the 38 in the literature. Ó 2016 The Author(s). Published by Elsevier Ltd on behalf of IBRO. This is an open access article under olfactory bulb the axons coalesce and form glomeruli 39 the CC BY-NC-ND license (http://creativecommons.org/ (Mombaerts et al., 1996; Feinstein et al., 2004; Feinstein 40 licenses/by-nc-nd/4.0/). and Mombaerts, 2004). 41 The widespread success of the gene-targeted 42 approach of the OR-IRES-marker design may have 43 Key words: olfactory receptor, olfactory sensory neuron, overshadowed or diminished efforts to raise and 44 glomerulus, antibody, immunofluorescence, antigen retrieval. characterize OR antibodies. But these gene-targeted 45 8 mutations are designed to visualize the OSNs (including 46 their cilia, dendrite, cell body, axon, and axon terminals) 47 that express a given OR, and do not enable the 48 visualization of the subcellular localization of the OR 49 protein within the OSNs. An attempt at creating an OR: 50 *Corresponding author. GFP C-terminal fusion protein by gene targeting 51 E-mail addresses: [email protected] (V. F. Low), peter.mom- resulted, for reasons that remain unclear, in altered 52 [email protected] (P. Mombaerts). Abbreviations: aa, amino acid; AR, antigen retrieval; EDTA, positions of the corresponding glomeruli in the olfactory 53 ethylenediaminetetraacetic acid; GFP, green fluorescent protein; bulb (Feinstein et al., 2004). 54 IRES, internal ribosome entry site; MOE, main olfactory epithelium; Antibodies against mouse ORs revealed that OR 55 OMP, olfactory marker protein; OR, odorant receptor; OSN, olfactory 56 sensory neuron; PBS, phosphate-buffered saline; PFA, proteins are present not only in OSN cilia, dendrite, and paraformaldehyde; SDS, sodium dodecyl sulfate. cell body but also within the axon and axon terminals 57 http://dx.doi.org/10.1016/j.neuroscience.2016.12.044 0306-4522/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/). 1 NSC 17525 No. of Pages 11 7 January 2017 2 V. F. Low, P. Mombaerts / Neuroscience xxx (2017) xxx–xxx 58 (Barnea et al., 2004; Strotmann et al., 2004), thus sup- 5 mg/kg body weight, respectively) and perfused with 115 59 porting the notion that the OR protein is involved in axonal 5 ml ice-cold phosphate-buffered saline (PBS), followed 116 60 wiring of OSNs. by 15 ml 2% paraformaldehyde in PBS (PFA). The 117 61 A quarter of a century after the discovery of mouse heads were dissected, postfixed in 2% PFA for 118 62 mammalian OR genes (Buck and Axel, 1991), there are 2 h at 4 °C, and decalcified in 0.45 M EDTA in PBS 119 63 still few papers on immunofluorescence staining of the overnight at 4 °C. Samples were cryoprotected in, 120 64 mouse MOE and olfactory bulb with OR antibodies that successively, 15% and 30% sucrose in PBS at 4 °C, 121 65 are commercially available. There are numerous antibod- frozen in O.C.T. Compound (Tissue-Tek), sectioned at 122 66 ies against ORs on the market, but they remain largely 12 lm with a Leica CM3500 cryostat, and collected onto 123 67 uncharacterized. The signal yielded by such antibodies glass slides. Alternative fixation methods were also 124 68 can be affected by the high sequence homology between tested; perfusion with 4% PFA resulted in poorer 125 69 ORs, by post-translational modifications, by protein fold- antigenicity, and immersion fixation in 2% PFA for 2 h 126 70 ing, by protein–protein interactions, and by fixation without perfusion did not yield improved antigenicity. 127 71 crosslinking. To increase the accessibility of the antigen 72 to the antibody, antigen retrieval can be used to break Antigen retrieval. Various antigen retrieval steps were 128 73 some of the crosslinks formed during fixation and to alter tested to optimize visualization of the target protein. 129 74 the protein re-folding following heat denaturation (Shi These included a 10-min incubation in 2 M HCl at room 130 75 et al., 2001; Emoto, 2005). Epitope unmasking through temperature, or 10 min in ice-cold 100% methanol 131 76 antigen retrieval can allow for a more accurate detection, followed by 0.5% sodium dodecyl sulfate (SDS) in PBS 132 77 and therefore representation, of the antigen. at room temperature. Antigen retrieval with a pressure 133 78 Here, we describe our screening of 40 commercially cooker (2100 Retriever; BioVendor) was carried out with 134 79 available antibodies against human and mouse ORs in 10 mM citric acid pH 6.0 and 10 mM Tris pH 9.0 with or 135 80 the mouse MOE using various antigen retrieval without 0.05% Tween 20 and 2 mM EDTA. After heating 136 81 techniques, in coronal 12-lm sections of the MOE of in a pressure cooker to 121 °C, slides were left to cool 137 82 21- day-old C57BL/6J mice. We obtained reliable for 2 h in the retrieval buffer. Additionally, PBS washes 138 83 immunoreactive signals with two antibodies against were tested with 0.05% Tween 20 or 0.2% Triton X-100, 139 84 human ORs (due to presumed crossreactivity with and incubation with antibody was tested for various 140 85 mouse ORs) and six antibodies against mouse ORs in times during a period of 1–4 days, with and without 141 86 the mouse MOE. Of these six, two antibodies also 0.1% Triton X-100. Best results were obtained with PBS 142 87 stained specific glomeruli in the olfactory bulb. washes and incubation with antibody diluted with 0.1% 143 Triton X-100. There was no obvious difference in 144 88 EXPERIMENTAL PROCEDURES antigen detection with different incubation times.