Methyl methacrylate embedding to study the morphology and immunohistochemistry of adult guinea pig and mouse cochleae

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Contents lists available at ScienceDirect

Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth

Basic neuroscience

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Methyl methacrylate embedding to study the morphology and immunohistochemistry of adult guinea pig and mouse cochleae

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Peter Bako a,b , Mohamed Bassiouni a , Andreas Eckhard a,c , Imre Gerlinger b , Claudia Frick a , Hubert Löwenheim a,d , Marcus Müller a,d,∗

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Tübingen Hearing Research Centre, Department of Otolaryngology, Head and Neck Surgery, Eberhard Karls University Tübingen, Tübingen, Germany Department of Otorhinolaryngology and Head and Neck Surgery, University of Pécs, Pécs, Hungary c Massachusetts Eye and Ear Infirmary, Eaton-Peabody Laboratories, Harvard Medical School, Boston, MA, USA d Department of Otolaryngology, Head and Neck Surgery, Carl von Ossietzky University Oldenburg, Oldenburg, Germany b

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h i g h l i g h t s • • • •

Technovit 9100 New® , a low temperature embedding system was used on the inner ear. Preservation of the morphology and maintenance of the antigenicity was tested. The embedding system provided highly preserved morphology and immunogenicity. The embedding system allowed precise identification of specific cell types in the inner ear.

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a b s t r a c t

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Article history: Received 4 June 2015 Received in revised form 16 July 2015 Accepted 19 July 2015 Available online xxx

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Keywords: Cochlea Immunohistochemistry Morphology Organ of Corti Resin embedding Spiral ganglion neuron

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1. Introduction

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Background: Histological analysis of the cochlea is required to understand the physiological and pathological processes in the inner ear. In the past, many embedding techniques have been tested in the cochlea to find an optimal protocol that gives both good morphological and immunohistochemical results. Resins provide high quality cochlear morphology with reduced immunogenicity due to the higher polymerization temperature. New method: We used Technovit 9100 New® , a low temperature embedding system based on methyl methacrylate, on adult guinea pig and mouse cochleae to evaluate preservation of the morphology and maintenance of the antigenicity. Results: Conventional toluidine blue staining, as well as immunohistochemical staining with a set of commonly used antibodies, showed highly preserved morphology and immunogenicity of decalcified adult guinea pig and mouse cochleae. Comparison with existing method(s): We demonstrate both, well-preserved morphology and preservation of antigenicity, superior to other embedding techniques. Conclusions: Our results showed that the Technovit 9100 New® embedding system provided highly preserved morphology and immunogenicity with our protocol in adult guinea pig and mouse cochleae. © 2015 Published by Elsevier B.V.

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The cochlear anatomy is very delicate, and the fragile epithelium of the cochlear duct consists of cells of different sizes and shapes surrounded by fluid-filled spaces. The cochlea is embedded in the

∗ Corresponding author at: Universitäts-Hals-Nasen-Ohren-Klinik, Department of Otorhinolaryngology, Head & Neck Surgery, Elfriede-Aulhorn-Str. 5, 72076 Tübingen, Germany. Tel.: +49 7071 2988178; fax: +49 7071 293311. E-mail address: [email protected] (M. Müller).

hardest bony capsule in vertebrates. High quality cochlear histological analysis is essential to comprehend cochlear physiology and pathophysiology. Standard light microscopy utilizing classic stains such as toluidine blue or hematoxylin-eosin on paraffin or resin sections is routinely used for morphological studies. These techniques provide homogeneous tissue blocks during embedding by complete infiltration of the media in the cochlear structure. Additional information on the distribution and localization of specific proteins can be gained using immunohistochemistry. The epitopes must be accessible to the antibodies for protein detection. Although many attempts have been made in the past to improve the

http://dx.doi.org/10.1016/j.jneumeth.2015.07.017 0165-0270/© 2015 Published by Elsevier B.V.

Please cite this article in press as: Bako P, et al. Methyl methacrylate embedding to study the morphology and immunohistochemistry of adult guinea pig and mouse cochleae. J Neurosci Methods (2015), http://dx.doi.org/10.1016/j.jneumeth.2015.07.017

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Table 1 Contents of different solutions of the Technovit 9100 New® embedding system. Name of solution

Base solution (BS)

Pre-infiltration 2 Pre-infiltration 3 Infiltration Stock solution A Stock solution B

Stabilized; 200 ml Destabilized; 200 ml Destabilized; ad 250 ml Destabilized; ad 500 ml Destabilized; ad 50 ml

Polymethylmethacrylate (PMMA) powder

Dibenzoylperoxide (hardener 1)

20 g 80 g

1g 1g 1g 4g

properties of the different media and embedding protocols, highquality preservation of both the morphology and immunogenicity in the same tissue remains a challenge. 51 Paraffin embedding is widely used in morphological studies 52 (Gillespie et al., 2003; Scheper et al., 2009), but the high melting 53 temperature of paraffin causes heat-induced trauma that disrupts 54 the morphology (Merchant et al., 2006). Celloidin (O’Malley et al., 55 2009) provides high-quality morphologically preserved sections. 56 Application of this technique to immunohistology is limited due 57 to the incomplete removal of celloidin from the tissue (Merchant 58 et al., 2006). 59 For light or electron microscopy, resin embedding serves as 60 an optimal technique (De Groot et al., 1987; Shepherd et al., 61 1993; Glueckert et al., 2008), providing highly preserved anatomi62 cal structures even in semi-thin sections. Unfortunately, there is 63 limited use for immunohistology due to the high temperatures 64 65Q4 required for tissue polymerization (Ruddell, 1967). Low temperature polymerization embedding resin media, i.e., Lowicryl K4M, 66 has been used on rat cochlea (Hirt et al., 2010). However, the lack 67 of the complete removal of this resin results in limited applica68 tions for immunohistochemistry. To enhance the immunogenicity 69 of samples embedded in paraffin or resin, retrieval techniques are 70 widely used. High temperature antigen retrieval in different solu71 tions (Shi et al., 2011; Yamashita and Okada, 2014) indeed leads to 72 improved antigenicity, but it renders the protocol longer and more 73 complicated, and it is disadvantageous for tissue preservation. 74 Frozen sections are used as a gold standard in immunohistology. 75 Although many studies have attempted to improve tissue preser76 vation (Coleman et al., 2009), the quality of cryosections usually 77 remains inferior to the quality resulting from the above-mentioned 78 embedding techniques, mainly due to freezing artifacts and crystal 79 formation (Whitlon et al., 2001). 80 Technovit 9100 New® is a resin embedding system that contains 81 methyl methacrylate and catalysators, i.e., N, N-dimethylaniline 82 and benzoyl peroxide. This combination allows the medium to 83 polymerize at low temperatures, that is, between −8 ◦ C and −20 ◦ C, 84 which results in highly preserved integrity of the anatomy as well 85 86Q5 as immunoreactivity of bone sections (Yang et al., 2003). In our study, the applicability of the Technovit 9100 New® 87 embedding system was tested. Adult guinea pig and mouse 88 cochleae were chosen to show the reliability of the method 89 in parallel on species widely used in the field of hearing 90 research. We demonstrate well-preserved morphology with con91 ventional toluidine blue staining and preservation of antigenicity 92 in immunohistologically stained sections using a set of cochlear cell 93 type-specific antibodies without retrieval. 94 49 50

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2. Materials and methods

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2.1. Animals

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Seven adult guinea pig (BFA bunt; 8–34 weeks old) and 6 mouse (NMRI; 8 weeks old) cochleae were used. All animal procedures were approved by the authorities of the Regional Council

N,N,3,5-tetramethylaniline (hardener 2)

1-Decanthiol (regulator)

4 ml

2 ml

(Regierungspräsidium) of Tübingen (Reference no. HN 2/11 and 2012.11.06). 2.2. Tissue preparation For guinea pigs, general anesthesia with a combination of fentanyl citrate (0.025 mg/kg; Fentadon, Eurovet Animal Health), midazolam (0.2 mg/kg; Midazolam-Ratiopharm, Ratiopharm) and medetomidine hydrochloride (Sedator, 1 mg/kg; Eurovet Animal Health) was induced. In the guinea pigs, a 0.5 ml solution of embutramide, mebezonium iodide and tetracaine hydrochloride (T61; Intervet Deutschland GmbH, Unterschleißheim, Germany) was injected intracardially. The chest was opened, and perfusion with 4% phosphate buffered formaldehyde (Roti-Histofix, Roth, Karlsruhe, Germany) was performed through the opened left ventricle with a pressure of 120–140 mm of mercury controlled by a manual manometer. Mice were suffocated with carbon dioxide (CO2 ) and decapitated. The skull was quickly opened along the sagittal suture, and the complete bony labyrinth capsule was removed. In both species, to open the perilymphatic space of the cochlea, the apex was gently opened with a 27 G needle, stapes and the round window membrane were removed with forceps and the cochlea was perfused with cold 4% formaldehyde (Roti-Histofix) through the round window and postfixed for 2 h at 4 ◦ C. The cochleae were then stored in 1% Roti-Histofix at 4 ◦ C overnight. The temporal bones of guinea pigs were then thinned with a diamond burr. Decalcification (Sigma-Aldrich, St. Louis, MO, USA; pH: 7.4) of the mouse and guinea pig cochleae with 0.2 M EDTA was performed for 3 days and 4–6 days, respectively. 2.3. Embedding procedure Technovit 9100 New® base solution (regarded as stabilized base solution) was destabilized using a chromatography column filled with 50 g of Al2 O3 (Sigma-Aldrich, St. Louis, MO, USA). Solutions for the phases of pre-infiltration were prepared according to the guidelines of the manufacturer (Sigma-Aldrich). One gram of dibenzoylperoxide (hardener 1) was added to 200 ml of stabilized and destabilized base solution for the pre-infiltration 2 and pre-infiltration 3 solutions, respectively. Infiltration solution and stock solution A for polymerization (Table 1) were prepared in two steps as described by Steiniger et al. (2013). For the infiltra- Q6 tion solution, 20 mg of polymethylmethacrylate powder (PMMA powder) was diluted in 160 ml of destabilized base solution. As the powder did not dissolve easily, stirring for 3 h was necessary to obtain a homogenous solution. One gram of hardener 1 was then added, and base solution was added until the total volume reached 250 ml; the solution was then stirred for 1 h. For stock solution A for polymerization, the same procedure was performed with 80 mg PMMA powder in 300 ml of destabilized base solution, and then 4 g of hardener 1 was added. Base solution was added until the total volume reached 500 ml. For polymerization stock solution B, 4 ml of N,N,3,5-trimethylaniline (hardener 2) and 2 ml of 1-decanthiol (regulator) were diluted in a destabilized base solution until the

Please cite this article in press as: Bako P, et al. Methyl methacrylate embedding to study the morphology and immunohistochemistry of adult guinea pig and mouse cochleae. J Neurosci Methods (2015), http://dx.doi.org/10.1016/j.jneumeth.2015.07.017

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Table 2 Solutions, temperatures and incubation times used in the different phases of the Technovit 9100 New® embedding system. RT: room temperature, h: hour, d: day. Phase

Solution

Temperature

Incubation time

Dehydration

1. 30% Ethanol 2. 40% Ethanol 3. 50% Ethanol 4. 60% Ethanol 5. 70% Ethanol 6. 80% Ethanol 7. 90% Ethanol 8. 96% Ethanol 9. 100% Ethanol Xylene 1. Stabilized BS + xylene 1:1 2. Stabilized BS + hardener 1 3. Destabilized BS + hardener 1 Destabilized BS + hardener 1 + PMMA powder Destabilized BS + hardener 1 + PMMA powder: destabilized BS + hardener 2 + regulator (9:1)

RT RT RT RT RT RT RT RT RT RT RT RT 4 ◦C 4 ◦C −12 ◦ C

1h 1h 1h 1h Overnight 2×1h 2×1h 2×1h 2×1h 3×1h Overnight 8h Overnight 24 h 4d

Intermedium Pre-infiltration

Infiltration Polymerization

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total volume reached 50 ml. All solutions were prepared, aliquoted in glass vials and stored at −20 ◦ C for up to 6 months. After decalcification, the cochleae were dehydrated in a graded series of alcohol (30%, 40%, 50% and 60% for 1 h, 70% overnight, 80%, 90%, 96% and 100% ethanol two times for 1 h) followed by an intermedium phase with xylene (BDH Prolabo-VWR Chemicals, Dublin, Ireland) 3 times for 1 h (Table 2). Pre-infiltration of the tissue was performed in 3 steps: (1) stabilization with Technovit 9100 New® base solution with a 1:1 mixture of xylene overnight, followed by (2) 8 h incubation with stabilized pre-infiltration 2 solution, and then (3) overnight incubation with a destabilized base solution with dibenzoylperoxide (pre-infiltration 3 solution). For infiltration, a destabilized base solution with dibenzoylperoxide mixed with PMMA-powder was used for 1 day. Dehydration, intermedium and the first 2 preinfiltration phases were performed at room temperature, and the third pre-infiltration and the infiltration phases were performed at 4 ◦ C. A total of 9 ml of destabilized base solution with dibenzoylperoxide and polymethylmethacrylate (stock solution A) and 1 ml of destabilized base solution containing N,N,3,5-trimethylaniline and 1-decanthiol (stock solution B) were mixed for the polymerization of the tissue. A plastic mold with a cradle insert form, available from the manufacturer (Heraeus-Kulzer, Wehrheim, Germany; order number [o.n.] of the mold: 66009903; o.n. of cradle insert form: 64708955), was filled with 2 ml of the mixture of stock solutions A and B, and the cochlea was orientated within the mold with the round window facing down to section the cochlea in the midmodiolar plane. The mold was then completely filled with the mixture of stock solutions A and B. An exsiccator stored at 4 ◦ C was used to evacuate the solution for 20 min. Then, the mold was closed with its cover and stored at −12 ◦ C for polymerization for 4 days. 2.4. Coating of slides

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Slides (Superfrost Plus, R. Langenbrinck, Emmerdingen, Germany) were precoated with a 2:1 mixture of 2% Ponal Classic (Henkel, Düsseldorf, Germany) and 0.01% Poly-l-lysine (SigmaAldrich, St. Louis, MO, USA) for 15 min and left to dry in a standing position.

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Seven micrometer thick sections were cut with a microtome (Microm HM 355 S, Thermo Fisher Scientific, Waltham, MA, USA) that used a “D” knife and were placed on the precoated slides. To plane the sections, drops of 60% ethanol were placed on the tops of

the slides. Finally, the slides were covered with polyethylene films (Heraeus-Kulzer, Wehrheim, Germany; order number: 64712818) and gently squeezed with an object holder press (Heraeus-Kulzer, Wehrheim, Germany; order number: 64712819) overnight at 50 ◦ C. 2.6. Staining 2.6.1. Epoxy tissue staining To illustrate the preservation of the anatomical structures, midmodiolar sections were stained with 1:5 water-diluted ETS (Epoxy Tissue Stain, contains toluidine blue and basic fuchsin, Electron Microscopy Sciences, Hatfield, PA, USA) for 6 min at 42 ◦ C. The slides were then rinsed with distilled water, differentiated with 70% ethanol for 15 s and rinsed with water. After drying, the slides were cleared in xylene 3 times for 10 min, mounted with Pertex (Medito, Burgdorf, Germany) and covered with a cover slip (R. Langenbrinck, Emmerdingen, Germany). 2.6.2. Immunohistochemical labeling Before immunohistochemical labeling, deplastification of the methyl methacrylate-embedded sections was required. For each phase of deplastification, slides were put in glass chambers filled with different solutions: (1) xylene was used 2 times for 20 min, followed by (2) 20 min of 2-methoxyethyl-acetate (MEA, Merck, Hohenbrunn, Germany), (3) acetone (Applichem, Darmstadt, Germany) 2 times for 5 min, and (4) distilled water rinse 2 times for 2 min. After performing a 1 M phosphate buffered saline rinse (PBS, Gibco-Life Technologies, Carlsbad, CA, USA) 3 times for 5 min, 1% normal donkey serum (NDS, Sigma-Aldrich, St. Louis, MO, USA) in 0.2% Triton-X100 (Sigma-Aldrich, St. Louis, MO, USA) was used as a blockade buffer followed by incubation with primary antibodies in reaction buffer (0.5% NDS in 0.2% Triton-X100) overnight. The following primary antibodies were used: mouse-anti Neurofilament 200 (Sigma-Aldrich, St. Louis, MO, USA; dilution 1:400), goatanti Calretinin (Chemicon International, Hampshire, UK; dilution 1:400), goat-anti Sox2 (Santa Cruz Biotechnology, Dallas, Texas, USA; dilution 1:50), goat-anti Prestin (from Prof. Marlies Knipper, Tübingen; dilution 1:500), rabbit-anti Myosin7A (Axxora LLC, Farmingdale, NY, USA; dilution 1:400), and rabbit-anti Peripherin (from Dr. Annie Wolff, Paris; dilution 1:500). On the next day, after a 1 M PBS rinse 3 times for 5 min, secondary antibodies in reaction buffer were applied for 1 h. The following secondary antibodies were used: donkey-anti mouse Alexa 488 (Molecular Probes Inc.Life Technologies, Eugene, OR, USA; dilution 1:400), donkey-anti goat Alexa 594 (Molecular Probes Inc.-Life Technologies; dilution 1:400), and donkey-anti rabbit 546 and 647 (Molecular Probes

Please cite this article in press as: Bako P, et al. Methyl methacrylate embedding to study the morphology and immunohistochemistry of adult guinea pig and mouse cochleae. J Neurosci Methods (2015), http://dx.doi.org/10.1016/j.jneumeth.2015.07.017

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Table 3 Antibodies used for immunohistochemistry and immunofluorescence. Primary antibody

Distributor

No.

Concentration

Secondary antibody

Distributor

No.

Concentration

Ms-␣ NF 200 Gt-␣ calretinin Gt-␣ SOX2 Gt- ␣ prestin Rb-␣ myosin7A Rb-␣ peripherin

Sigma Chemicon St. Cruz Knipper Axxora Annie Wolf

N0142 AB1550 SC-17320

1:400 1:400 1:50 1:500 1:400 1:500

Dk ␣-ms Alexa Fluor 488 Dk ␣-gt Alexa Fluor 594 Dk ␣-gt Alexa Fluor 594 Dk ␣-gt Alexa Fluor 594 Dk ␣-rb Alexa Fluor 647 Dk ␣-rb Alexa Fluor 546

Molecular probes Molecular probes Molecular probes Molecular probes Molecular probes Molecular probes

A21202 A11058 A11058 A11058 A31573 A10040

1:400 1:400 1:400 1:400 1:400 1:400

PTS-25-6790

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Inc.-Life Technologies; dilution 1:400). All primary and secondary antibodies were diluted in 0.5% NDS in 0.2% Triton-X100 (reaction buffer) and are detailed in Table 3. After a PBS rinse 3 times for 5 min, diamidino-phenylindole staining (DAPI, Molecular Probes Inc.-Life Technologies, Eugene, OR, USA; 1:100 in PBS) was used for 10 min to visualize the nuclei. After a PBS rinse (2 times for 5 min), the slides were covered with FluorSave Reagent (Merck Millipore, Darmstadt, Germany), and a cover slip was added (R. Langenbrinck, Emmerdingen, Germany).

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2.7. Imaging

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All micrographs were taken using a Zeiss Axio Imager 2 (Zeiss, Oberkochen, Germany) equipped with an Apotome.2 unit (Zeiss) using ZEN 2012 software (Zeiss).

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In Fig. 1B, a midmodiolar image of an adult mouse cochlea with 4 half-turns is shown. Fig. 1A and B are shown with the same scale to illustrate the size difference of the two cochleae. In both images, an intact Reissner’s membrane at all half-turns of the midmodiolar sections is visible. Detailed morphological analysis is shown in Fig. 2. A cross section of the second half-turn of the guinea pig cochlea is illustrated in Fig. 2A. Scala vestibuli (SV), scala media (SM) and scala tympani (ST) showed integrity; the Reissner’s membrane (black arrow) was extended. Epithelia of the SM were morphologically preserved, the

ETS staining demonstrated the morphological preservation of the Technovit 9100 New® embedded cochleae. A set of antibodies was used to stain cells of the organ of Corti and Rosenthal’s canal (RC). An overview of images of the adult guinea pig and mouse cochleae stained with ETS is shown in Fig. 1. The midmodiolar section of the guinea pig cochlea with 8 half-turns is shown in Fig. 1A.

Fig. 1. Midmodiolar section of adult guinea pig and mouse cochleae stained with ETS. (A) Midmodiolar section of the guinea pig cochlea with 8 half-turns. (B) A midmodiolar section of the mouse cochlea is shown with the same scale as in 1A, showing the difference in size between the two cochleae. Four half-turns are visible. Reissner’s membrane of all half-turns in both species is intact.

Fig. 2. ETS stained overview, organ of Corti and Rosenthal’s Canal image of the second half turn of adult guinea pig and mouse cochleae. (A) Cross section of the second halfturn of the guinea pig cochlea. Scalae (SV, ST, SM) are intact. The SL is attached tightly to the bony wall, Reissner’s membrane (black arrow) is intact and stretched, and the organ of Corti is connected to the basilar membrane without any separation. The preserved morphology of the peripheral processes of the SGNs in the OSL and their perikarya in RC is visible. (B) Overview of the mouse cochlear second halfturn. Reissner’s membrane (black arrow) is preserved but crooked; the organ of Corti is intact and attached tightly to the basilar membrane. White arrows show a slight dissection of the SL from the lateral bony wall. (C) and (D) Organ of Corti of the second half turn of the guinea pig (C) and the mouse cochlea (D). Images show highly preserved morphology in both species. Hair cells can be distinguished from their supporting cells, Pillar cells are intact, and stereocilia in OHCs and IHC are preserved in guinea pig. (E) and (F) RC of guinea pig (E) and mouse (F) cochleae. In both species, nuclei and nucleoli of the SGNs can be recognized; cells are well separated from each other. The intraganglionic spiral bundle (white arrow) in guinea pig is also visible. The preserved attachment between the peripheral cells and a bony border of RC can also be observed in both species. Scale bar: (A) and (B) 50 ␮m. (C)–(E) 20 ␮m.

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Fig. 4. Staining pattern of prestin and calretinin antibodies in the guinea pig (A and C) and mouse organs of Corti (B and D). (A and B): Prestin (red) antibody labels all 3 OHCs lateral plasma membranes in both species (labeled with white stars). IHCs remain unstained (white arrows). (C) In the guinea pig organ of Corti, IHCs (white arrow) as well as the 3 rows of the Deiters cells (white arrowhead) are stained with calretinin (violet). Labeling of the processes of the Deiters cells is also visible. (D) In mouse, only IHCs show immunoreactivity (white arrow) with calretinin, while Deiters cells remain unstained. Peripheral processes of the SGNs are labeled below the IHCs (empty white arrow) in guinea pig as well as in mouse specimens. In both species, OHCs are unstained (white stars). Scale bar: 20 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Immunohistochemistry images of the second half-turn in guinea pig (A, C, E, G, and I) and mouse (B, D, F, H and J) organs of Corti. (A and B) Merged image of Myosin7A (red), Sox2 (orange) and NF200 (green) staining with DIC and DAPI (blue). (C and D) Myosin7A (red) labels the cytoplasm of 1 IHC and 3 OHCs in both species. The basilar membrane also shows immunoreaction ((C) arrows). (E and F). Nuclear staining with Sox2 (orange) antibodies. All 3 Deiters cells, 2 Hensen cells, and the inner and outer pillar cells are labeled in guinea pig. No labeling of the border cell medial to the inner hair cell is visible. In mouse, a border cell is labeled, but 1 Hensen cell and the outer pillar cell remain unstained. In these sections, 2 inner phalangeal cells are visible in both species, and all of them are labeled. (G and H) NF200 (green) labels the peripheral processes of the SGNs. Staining of a nerve fiber crossing the Corti tunnel in guinea pig is visible, but its destination to the OHCs could not be observed. (I and J). DAPI staining. Scale bar: 20 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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spiral ligament (SL) and bony lateral wall were tightly connected, and the organ of Corti laid firmly on the basilar membrane. Peripheral processes of the spiral ganglion neurons (SGN) in the osseous spiral lamina (OSL) and their perikarya in RC showed highly preserved morphology. In Fig. 2B, the second half-turn of the mouse cochlea is demonstrated. The SL was slightly separated from the bony wall (labeled with arrows) in the upper part of the SM. Reissner’s membrane was crooked but intact (black arrow). The cell membranes of SGNs laying in RC were intact, and nuclei could be distinguished from the cytoplasm. In Fig. 2C and D, the second halfturn organ of Corti are illustrated. The three images of outer hair cells (OHCs) and one of inner hair cell (IHC) could be recognized and

distinguished from their supporting cells. Pillar cells were intact in both species. In the guinea pig image (Fig. 2C), the stereocilia of the hair cells could be observed, and the Corti tunnel showed preservation. In Fig. 2E and F, the preserved structure of the RC is illustrated. SGNs could be distinguished from each other, and nuclei and nucleoli were recognizable in both species. In guinea pig, the intraganglionic spiral bundle (white arrow) was visible. The intact bony border of RC was demonstrated in both species. Immunofluorescence staining of the second half-turn of the adult guinea pig (Fig. 3A, C, E, G and I) and mouse (Fig. 3B, D, F, H and J) organs of Corti with Myosin7A, Sox2 and NF200 is shown in Fig. 3. Merged images of immunolabeling with differential interference contrast (DIC) are shown in Fig. 3A and B. Co-staining using three antibodies on deplastified guinea pig and mouse sections was produced. Myosin 7A (Fig. 3C and D, red) stained the cytoplasm of 3 OHCs and the 1 IHC in both species. In guinea pig, the basilar membrane (arrows) also showed staining with the same intensity as the hair cell labeling. Sox2 (Fig. 3E and F, orange) labeled the nuclei of all 3 Deiters cells and 2 Hensen cells as well as the inner and outer pillar cells in guinea pig (Fig. 3C), although the staining of the lateral Hensen cell was weaker. No staining of the border cell medial to the inner hair cell could be observed. In mouse (Fig. 3D), 3 Deiters cells, 1 Hensen cell, the inner pillar cell and as well as a border cell showed positive immunolabeling, but 1 Hensen cell at the edge of the image as well as the outer pillar cell remained unstained. In these sections, 2 inner phalangeal cells were present in both species, whose nuclei were also labeled with Sox2. NF200 (Fig. 3G and H, green) stained the peripheral processes of the SGNs. In guinea pig, nerve fibers crossing the Corti tunnel were also visualized, but the destination to the OHC could not be observed. DAPI staining of the nuclei is shown in Fig. 3I and J. Fig. 4 shows labeling patterns of the organ of Corti stained with prestin and calretinin antibodies. These antibodies were chosen to demonstrate their known staining patterns in the two types of hair cells. Prestin is a motor protein of the OHC (Zheng et al., 2000) and is expressed exclusively in this cell type in the organ of Corti.

Please cite this article in press as: Bako P, et al. Methyl methacrylate embedding to study the morphology and immunohistochemistry of adult guinea pig and mouse cochleae. J Neurosci Methods (2015), http://dx.doi.org/10.1016/j.jneumeth.2015.07.017

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Fig. 5. Results of immunostaining of Rosenthal’s Canal in guinea pig (A, B, E, F, I and J) and mouse (C, D, G, H, K and L) at two magnifications (lower [A, C, E, G, I and K] and higher [B, D, F, H, J and L]). The latter images are subsections of the lower magnification images illustrated with the white boxes. (A and B). Calretinin staining in guinea pig cochlea. Peripheral (white arrow) as well as central processes (hollow white arrow) and cell bodies of the SGNs showed labeling with calretinin. Nerve fibers in the modiolus were also labeled (star). The intensity of the staining of the SGN cell body varied. Not all of the cells were labeled with calretinin (marked with white arrowheads), but the stained cells showed both cytoplasmic and nuclear positivity. (C and D) In the mouse cochlea, the same pattern of staining was observed with completely unlabeled SGNs (white arrowheads). (E and F) Peripherin staining of the guinea pig RC. All SGN cell bodies showed labeling with diverse intensity. One SGN cell body showed a highly intense signal (white arrow in (F)) (G and H) In the mouse section, only 4 cell bodies with eccentric nuclei were labeled with peripherin (white arrows). These cells were located in the lateral part of RC, and 3 of them formed a cluster. (I and J) NF200 staining in guinea pig. Peripheral (white arrows) as well as central processes (hollow white arrows) of the SGNs were observed. Modiolar nerve fibers were also labeled (white star). Cytoplasmic labeling of all SGN cells is visible, as are the nerve fibers inside the intraganglionic spiral bundle (white star). (K and L) The same staining pattern is demonstrated in mouse RC. Peripheral (white arrows) and central processes (hollow white arrows) as well as all SGN cell bodies showed NF200 labeling. The stained central processes are collected in the modiolus (white star). Scale bar: 20 ␮m.

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Conversely, calretinin is expressed in IHCs but not in OHCs (Pack and Slepecky, 1995). In our study, prestin (Fig. 4A and B) antibodies labeled the lateral wall of the 3 OHCs in both species (white stars). Labeling was restricted to the lateral plasma membrane. No staining of the IHCs was observed (white arrows). Calretinin stained the 3 rows of Deiters cells (white arrowheads) as well as the IHCs (white arrow) in guinea pig (Fig. 4C). Processes of the Deiters cells were also stained. In the adult mouse cochlea, only IHCs (white arrow) showed specific staining, and the Deiters cells remained unstained (Fig. 4D). Labeling of peripheral processes of SGNs (hollow white arrow) was observed in both species. In Fig. 5, labeling patterns using antibodies against calretinin (Fig. 5A–D), peripherin (Fig. 5E–H) and NF200 (Fig. 5I–L) of the SGNs are shown. Images at lower magnification illustrate the guinea pig (Fig. 5A, E and I) and mouse (Fig. 5C, G and K) RCs. For detailed visualization of the SGN cell body staining pattern, higher magnification images are shown for both species in Fig. 5B, D, F, H, J and L. These images are shown in the lower magnification images and illustrated with white boxes. In guinea pig, peripheral (white arrow) as well as central processes (hollow white arrow) and cell bodies of the SGN showed labeling with calretinin (Fig. 5A). Nerve fibers in the modiolus were also labeled (white arrowhead). In Fig. 5B, the staining pattern of the SGN cell bodies is shown. The staining intensity varied among the SGNs. Not all of the cells were labeled with calretinin (unmarked examples with white arrowheads), but the stained cells showed both cytoplasmic and nuclear positivity. The same pattern

of SGN cell body staining was observed in mouse SGNs (Fig. 5C and D). In Fig. 5D, unstained cell bodies are marked with white arrowheads. Peripherin stained all ganglion neurons in guinea pig (Fig. 5E and F) where cytoplasmic as well as nuclear staining could be observed. One of the SGNs (white arrowhead) showed more intense staining. (Fig. 5F). In mouse (Fig. 5G and H), only 4 cell bodies with eccentric nuclei were labeled with peripherin (white arrows). These cells were located in the lateral part of RC, and 3 of them formed a cluster. NF200 labeling is shown in Fig. 5I–L. In the low magnification images (Fig. 5I and K), staining of the central processes (hollow white arrows) of the SGNs could be observed, and modiolar nerve fibers were also labeled (white star). In the high magnification images (Fig. 5J and L), cytoplasmic staining against NF200 could be seen in all SGNs. The labeled peripheral processes of the cells are marked with white arrows. In the guinea pig image (Fig. 5J), nerve fibers in the intraganglionic spiral bundle (white star) were stained. 4. Discussion In the past, many attempts have been made to establish an optimized histological protocol that preserves both the morphology and immunogenicity of the cochlea. Properties of the embedding material, such as the melting temperature and polymerization, as well as freezing artifacts usually limited the applicability of the technique for both purposes. In this study, we tested a methyl

Please cite this article in press as: Bako P, et al. Methyl methacrylate embedding to study the morphology and immunohistochemistry of adult guinea pig and mouse cochleae. J Neurosci Methods (2015), http://dx.doi.org/10.1016/j.jneumeth.2015.07.017

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methacrylate-based embedding system, Technovit 9100 New® , on adult guinea pig and mouse cochleae. 4.1. Preservation of morphology Other studies that focused on preservation of both morphology and immunogenicity examined various cochlear structures to evaluate the quality of their technique. Coleman et al. (2009) showed the preserved integrity of the organ of Corti and visualized the nuclei of SGNs in cryosections. In gelatin-embedded sections, the integrity of the scalar walls, Reissner’s and basilar membranes, hair cells, peripheral and central processes and perikarya of the SGNs were used as indicators of the preservation (Hurley et al., 2003). O’Malley et al. (2009) compared sections embedded in paraffin, polyester wax and celloidin and examined the preservation of Reissner’s and basilar membranes and the cells of the cochlear duct and found that the best preservation occurred in celloidin sections. We observed well-preserved morphology and maintained integrity of the scalae and Reissner’s membrane in both species. The OSL containing the peripheral processes of the SGNs was intact (Fig. 2A and B), and SGN perikarya showed that preservation allows the identification of the nucleoli of the cells (Fig. 2E and F). Maintained anatomy of the organ of Corti was demonstrated in high magnification images, and various cells could be distinguished both in guinea pig and in mouse (Fig. 2C and D). 4.2. Preservation of immunogenicity

We tested specific inner ear cell markers on Technovit 396 9100 New® -embedded sections. All hair cells were stained with 397 myosin7A (Fig. 3C and D). NF200 labeling for SGNs, both for the 398 cell body and their peripheral and central processes, is commonly 399 reported in the literature (Hafidi, 1998; Bailey and Green, 2014), 400 which we could also reproduce in our sections (Fig. 5I–L). Peripheral 401 nerve fibers could be followed to the IHCs in both species. Further402 more, fibers crossing the organ of Corti tunnel were labeled (Fig. 3G 403 and H). Sox2 expression was shown in the nuclei of all subtypes of 404 supporting cells in adult mouse cochleae (Oesterle and Campbell, 405 Q7 2009). Our findings showed a similar pattern (Fig. 3E and F). 406 In the literature, diverse expression patterns of calretinin in the 407 organ of Corti in guinea pig and mouse have been reported. In 408 guinea pig, Pack and Slepecky reported calretinin positivity in IHCs 409 and also in Deiters cells in plastic-embedded sections. Labeling of 410 Hensen cells was also illustrated in cryosections (Dechesne et al., 411 Q8 1992). In the adult mouse, supporting cells remained unstained, 412 and only labeling of IHCs could be observed (Dechesne et al., 1994). 413 We found IHC immunolabeling in both species, and Deiters cells 414 were positive for calretinin only in guinea pig (Fig. 3C and D). 415 Peripherin, an intermediate filament protein, has been widely 416 used as a specific marker of the type II SGN in mouse and rat (Huang 417 et al., 2007; Hafidi et al., 1993). Some features of type II SGNs were 418 summarized by Hafidi (1998): They have an eccentric nucleus and 419 usually form a cluster of 2 or 3 cells located in the lateral part of 420 RC. Conversely, Liu et al. (2009) showed nuclear and cytoplasmic 421 labeling of peripherin in type I SGNs in pig. They showed 1 cell with 422 a brighter signal, which could be a type II neuron. We found differ423 ent staining patterns in the two examined species. In guinea pig, all 424 SGN cells appeared to be stained (Fig. 5E and F) with one showing 425 substantially more intense staining. In the presented mouse image, 426 a cluster of 3 cells and 1 single cell with an eccentric nucleus posi427 tion showed immunoreactivity (Fig. 5G and H). These cells were 428 located in the lateral part of RC, showing the same characteristics 429 Hafidi mentioned for SGN type II. 430 We demonstrated that Technovit 9100 New® , a low tem431 perature resin embedding system, provides highly preserved 395

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morphology and immunogenicity with our protocol of decalcified adult guinea pig and mouse cochleae. Parallel staining of adjacent sections from the same tissue block with conventional staining and immunohistochemistry allowed precise identification of specific cell types in the inner ear. It served as a valuable tool to investigate various processes, such hair cell loss or outcomes of pharmacological treatment. Uncited reference Dechesne et al. (1991). Acknowledgments

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442 The study was supported by the NanoCI project (project num443 ber: 281056) under the 7th Framework Program of the European Union. The first author received a grant from DAAD (German aca- Q10444 Q11 445 demic exchange service). Special thanks to Prof. Marlies Knipper 446 for the Prestin and to Dr. Annie Wolff for the Peripherin antibodies.

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