Immunohistochemical techniques for the human inner ear

Abstract

In this review, we provide a description of the recent methods used for immunohistochemical staining of the human inner ear using formalin-fixed frozen, paraffin and celloidin-embedded sections. We also show the application of these immunohistochemical methods in auditory and vestibular endorgans microdissected from the human temporal bone. We compare the advantages and disadvantages of immunohistochemistry (IHC) in the different types of embedding media. IHC in frozen and paraffin-embedded sections yields a robust immunoreactive signal. Both frozen and paraffin sections would be the best alternative in the case where celloidin-embedding technique is not available. IHC in whole endorgans yields excellent results and can be used when desiring to detect regional variations of protein expression in the sensory epithelia. One advantage of microdissection is that the tissue is processed immediately and IHC can be made within 1 week of temporal bone collection. A second advantage of microdissection is the excellent preservation of both morphology and antigenicity. Using celloidin-embedded inner ear sections, we were able to detect several antigens by IHC and immunofluorescence using antigen retrieval methods. These techniques, previously applied only in animal models, allow for the study of numerous important proteins expressed in the human temporal bone potentially opening up a new field for future human inner ear research.

Keywords: Antigen retrieval; Archival human temporal bone; Celloidin-embedded sections; Cochlea; Cryosections; Immunohistochemistry; Microdissection; Paraffin; Vestibule.

Fig. 1

Immunofluorescence (IF) staining in frozen sections obtained from microdissected auditory and vestibular endorgans. a Neurofilaments-IF in the organ of Corti (oc). Arrows point to IF terminals and fibers. Tunnel of Corti (tc), spiral limbus (sli), lumen (lu). b Neurofilament-IF in the spiral ganglia neurons arrows point to IF large-size spiral ganglia neurons, thick arrowhead point to IF small-size spiral ganglia neurons. c Na⁺K⁺ATPase-IF in satellite cells (arrowheads) that surround spiral ganglia neurons. d Smooth muscle actin-IF in a blood vessel of the modiolus. e Neurofilament-IF in nerve fibers located in the crista stroma (st) and underneath the sensory epithelia (se) (green color, arrows), phalloidin staining (red) at the apical portion of the sensory epithelia. f Collagen IV-IF in basement membrane (bm) of the utricle stroma. g Cx-26-IF in supporting cells (sc) of the utricle sensory epithelia. h Aquaporin-4-IF (red) and GFAP-IF (green) in the cytoplasm of supporting cells (sc) of the utricle sensory epithelia. Hair cells (hc) were non-IF. Arrows point to AQP4-IF (red) at the basolateral portion of the sc. i Kir 4.1-IR in the stria vascularis (stv) of the cochlea, spiral ligament (sl). j WARP-IF in blood vessels of the stria vascularis (stv), spiral ligament (sl), basilar membrane (b), Rosenthal’s canal (rc). k Cytochrome-C-IF in hair cells (arrows) of the sensory epithelia (se). l Cx30-IF in the crista ampullares sensory epithelia (se) and stroma (st) (red punctate) and l′ the cochlea, red punctate in the organ of Corti (oc), spiral ligament (sl). DAPI in blue color shows staining in cell nuclei. Magnification bars a 150 μm, b 125 μm, c 125 μm, d 50 μm, e 70 μm, f 50 μm, g 50 μm, h 25 μm, i 80 μm, j 200 μm, k 50 μm, l 125 μm, l′ 125 μm

Fig. 2

Immunohistochemical staining in paraffin sections obtained from microdissected endorgans. a Cochlin-IR in a cross section of the macula utricle. Cochlin-IR was seen in the basement membrane (bm) of the sensory epithelia and extracellular matrix of the stroma (st). The sensory epithelium (se) was non-IR. b Superoxide dismutase-2 (SOD-2)-IR in the cytoplasm of the vestibular ganglia neurons (vgn). c WARP-IR in a section of the vestibular ganglia. WARP-IR was located in blood vessels (bv). Vestibular ganglia neurons (vgn) and nerve fibers (nf) were non-IR. Bar 100 μm in every figure

Fig. 3

Immunofluorescence staining in whole-mount preparations. a Myosin VIIa in the mid-apical portion of the cochlea. Inner hair cells (ihc) and outer hair cells (ohc) were IF to myosin VIIa (red). b Double immunolabeling of myosin VIIa (red) and Na⁺K⁺ATPase (green). c The organ of Corti showing phalloidin Oregon green staining in the inner (ihc) and outer (ohc) hair cells stereocilia and the apical portion of Deiter’s cells (dc). Myosin VIIa-IF (red) was seen in the hair cell cytoplasm. d Neurofilament-IF in the whole cochlea, apical (a), middle (m), basal (b), hook region (h). e Higher magnification view of the apical portion of the cochlea in d. f Calmodulin-IF in hair cells of the macula saccule. g Neurofilament-IF in fibers of the macula utricle. Thick nerve fibers and calyceal terminals (arrows) are clearly identified. h Double labeling of myosin VIIa (red) and neurofilaments (green). Magnification bars a 400 μm, b 150 μm, c 50 μm, d 250 μm, e 100 μm, f, g 100 μm, h 150 μm

Fig. 4

IHC in celloidin-embedded sections. a β2-Laminin-IR within basement, vascular (arrows) and perineural membranes (dark amber color) in the stria vascularis (stv), spiral ligament (sl), organ of Corti (oc), Reissner’s membrane (rm), spiral limbus (sli), and the basilar membrane. The tectorial membrane (tm) was non-IR. a′ β2-Laminin-IR in perivascular basement membrane of the spiral ligament (sl) and stria vascularis (stv) (arrows). a″ β2-Laminin-IR in perivascular (long arrows) and perineural (short arrows) basement membranes of the spiral ganglia. Spiral ganglia neurons (sgn). a‴ β2-Laminin-IR in the basement membrane underneath the sensory epithelia (short arrows) and also perivascular (long arrows) basement membranes, hair cells (hc), lumen (lu). b Collagen IV-IR in stria vascularis (stv) and spiral ligament (sl) perivascular basement membranes (arrows), spiral prominence is also collagen IV-IR (sp). b′ Collagen IV-IR was seen at the spiral limbus (sli), organ of Corti (oc). b″ Collagen IV-IR in perineural (short arrow) and perivascular (arrows) basement membrane of the spiral ganglia. Spiral ganglia neurons (sgn). c SOD2-IR in the spiral ganglia neurons (sgn). d Neurofilaments-IR in the spiral ganglia neurons (sgn). e Acetylated tubulin-IR in the pillar cells (pc), and Deiter’s cells. e Celloidin-embedded tissue sections counterstained with hematoxylin (except for a′, d, e). Magnification bars a 200 μm, a′ 100 μm, a″ 100 μm, a‴ 100 μm, b 75 μm, b′ 75 μm, b″ 120 μm, c, d 150 μm, e 40 μm

Fig. 5

Immunofluorescence staining of prestin and acetylated (a)-tubulin in the organ of Corti. Celloidin-embedded section. a Prestin-IF (green) was seen in outer hair cells (ohc), acetylated tubulin (red) was seen in pillar cells (pc), Deiter’s cells (dc) and the cells of the spiral prominence (sp). Photomicrograph obtained with a fluorescent microscope. b Photomicrograph obtained with a laser confocal microscope as in (a higher magnification), prestin-IF was localized in outer hair cells (green), acetylated tubulin-IF identified in the Deiter’s cells (dc) and pillar cells (pc) (red). Magnification bars a 70 μm, b 15 μm