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.

Appendices

Appendix

Specimens

The Institutional Review Board (IRB) of UCLA approved this study (IRB #14-001753). Appropriate informed consent for inclusion in the study was obtained from each temporal bone donor. The temporal bone donors used in this study were part of a National Institute of Health funded Human Temporal Bone Consortium for Research Resource Enhancement. Auditory and vestibular endorgans were microdissected from postmortem temporal bones from seven subjects with a documented history of normal auditory and vestibular function. The vestibular endorgans and cochlea were microdissected from one temporal bone from each donor. Celloidin-embedded sections were selected from several temporal bones of our temporal bone bank collection. Table 2 shows the source of the specimens used.

Table 2 Specimens used for IHC (HRP-DAB) and IF

Microdissection of auditory and vestibular endorgans

At autopsy, the whole brain, including the brainstem and blood vessels, was removed from the cranial cavity. The 8th cranial nerve and vascular bundle were sectioned outside the internal auditory canal. The temporal bone was then removed as described by Schuknecht (1968) using a bone plug cutter. The bones were then immediately immersed for 16 h in ice-cold 10 % buffered formalin. Thereafter, the fixative was removed by washing with phosphate buffer (0.1 M, pH 7.2) PB (20 min × 3). Before the auditory and vestibular endorgan microdissection, the connective and muscle tissue was removed. The tympanic membrane and ossicles were then removed, and the temporal bones are immersed in 10 % EDTA (pH 7.4) for 3 days. Temporal bones immersed in EDTA were placed under a dissecting microscope (Nikon SMZ1500), and using forceps, the bone that surrounds the membranous labyrinth was carefully removed (Lopez et al. 2005a, 2007; Ishiyama et al. 2009). The oval window was identified, and surrounding bone was carefully removed until the utricle was visualized and removed; then, the horizontal and superior crista ampullaris were identified and removed. Thereafter, the saccule and posterior canal were also removed. The bone that surrounds the cochlea was further removed, and the remaining bone that contains the membranous portion of the cochlea was kept in 10 % EDTA for 1 week; then, the bone was carefully removed and the whole cochlea was detached from the bone. The microdissected auditory and vestibular endorgans were placed in 10 % EDTA for one more day, and cryostat sections were obtained.

Cryostat sections

The whole microdissected cochlea or vestibular endorgan was immersed in 30 % sucrose in PBS for 7 days for cryoprotection. The endorgans were removed from the sucrose solution and immersed in Tissue Tek^® compound (VWR). Before sectioning, the dissected vestibular endorgans and cochlea were placed on Teflon embedding molds (Polysciences Inc.) and properly oriented under the dissecting microscope to obtain longitudinal mid-modiolar sections of cochlea, and cross sections of the macula utricle or saccule and of the cristae ampullares. Endorgans were placed under vacuum for 4–6 h to allow infiltration of Tissue Tek. Twenty-micron-thick serial sections were obtained using a Microm-H cryostat (Microm-HN505E). Three consecutive sections were placed in each slide. By doing this step, up to six different antibodies can be simultaneously screened (one different monoclonal and one polyclonal antibody per tissue section) per slide. The cryostat sections were mounted on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA, USA) and stored at −80 °C until they were used. Cryostat sections properly stored can be used for IHC several years after being sectioned. Before that IHC sections were thawed and allowed to dry for 30 min. Next, with the aid of a pen that contains hydrophobic solution (ImmEdge pen, Vector) a line around each tissue section was made to prevent the mixture of the different solutions.

Immunofluorescence

After thawing at room temperature for 10 min, tissue sections were incubated at room temperature for 60 min with a blocking solution containing 1 % bovine serum albumin (BSA) fraction V (Sigma, St. Louis, MO, USA) and 0.5 % Triton X-100 (Sigma) in PBS. At the end of the incubation, the blocking solution was removed and the primary polyclonal or monoclonal antibody against the specific protein was applied (Table 3). Primary antibodies were incubated overnight at 4 °C in a humidity chamber. The secondary antibodies against rabbit or mouse labeled with Alexa 488 or 594 (1:1000, Molecular Probes, Carlsbad, CA, USA) were applied and incubated for 2 h at room temperature in the dark. At the end of the incubation, sections were washed with PBS (3 × 10 min) and covered with Vectashield mounting media containing DAPI (Vector Labs, Burlingame, CA, USA) to visualize all cell nuclei.

Table 3 Antibodies used in the present study

Antibodies

The characteristics and specificity of the antibodies used in this study have been described in detail (Duong et al. 2011; Ishiyama et al. 2009, 2010; Lopez et al. 2005a, 2007, 2009; Calzada et al. 2012a, b; Balaker et al. 2013; Ahmed et al. 2013; Nguyen et al. 2014; Vorasubin et al. 2016) (Table 3).

Controls

As positive controls, human kidney and brain (cerebral and cerebellar cortex) cryostat sections from the same subjects were used. These sections were subjected to the same protocol as the IF protocol of vestibular endorgans and cochlea cryostat sections. As negative controls, the primary antibody was omitted and the immunoreaction was performed as described above. In all cases, no immunoreaction was detected.

Quality of frozen sections obtained from microdissected endorgans

It is well known that longer post-mortem times are associated with poorer morphology as demonstrated on hematoxylin and eosin staining (Ishiyama et al. 2009). The immunoreactive pattern in the tissue from the temporal bones used in this, and previous studies give similar results.

IF staining in whole auditory and vestibular endorgans

The microdissected whole cristae, macula utricle, or cochlea was immersed in 30 % sucrose in PBS (0.1 M, pH 7.4) for 1 week. Individual endorgans were subjected to a freezing step that was accomplished after removing most of the sucrose solution from the container, leaving only enough solution to cover the tissue. For the freezing step, auditory or vestibular endorgans were placed in a plastic vial and immersed in liquid nitrogen and immediately thereafter removed, and the endorgans were left to thaw for 5 min and the sucrose solution replaced by PBS before IF staining (Lopez et al. 2005a).

Immunofluorescence (IF)

The endorgans were placed in a rotary shaker and incubated for 3 h in a blocking solution containing 2 % BSA fraction V (Sigma, SLM), 0.1 % TritonX-100 (Sigma, SLM) diluted in PBS at 4–6 °C. Subsequently, the blocking solution is removed and the whole organs are incubated for 48 h with the primary antibodies (Table 3), placing the vials in the rotatory shaker in a cold room. At the end of the incubation, the secondary antibodies against rabbit labeled with Alexa 594 were diluted 1:1000 in PBS (Molecular Probes, Eugene, OR, USA) or the secondary antibodies against mouse labeled with Alexa 488 (1:1000 in PBS, Molecular Probes) were applied to the tissue sections and were incubated for 1 h at room temperature in the dark. At the end of the incubation, the whole endorgans or sections were washed with PBS (20 min × 5) and mounted flat on glass slides with Vectashield solution containing DAPI (Vector Labs, Burlingame, CA, USA) and immediately coverslipped. The coverslip was attached to the glass slide using nail polish around the glass slide.

Phalloidin staining in whole endorgans

Following microdissection and permeabilization steps, vestibular endorgans were incubated in a blocking solution that contained 1 % BSA (Sigma, Fraction V) for 1 h and then phalloidin Oregon Green (20 units–10 µL, Molecular Probes) for 15 min. Following incubation at room temperature, the tissue was washed with PBS and mounted on glass slides as described above for IF.

IHC in paraffin-embedded tissue sections

After microdissection from the temporal bone, the cochlea and the vestibular endorgans were placed overnight in 10 % EDTA (in PB, pH 7.4) on a rotary shaker. The specimens were then washed twice in PB for 15 min. Specimens underwent dehydration by serial immersion for 5 min each in an increasing concentration of ethanol as follows: 35, 50, 70, 95 then 100 %. The specimens were then transferred to a 1:1 mixture of 100 % ethanol and 100 % xylene for 5 min and placed in the oven at 65 °C for 5 min. Using a wood stick, the endorgans were then transferred to melted paraffin and placed in the oven for 5 min at 60 °C. Specimens were placed in individual Teflon-coated silicon molds (Polysciences) filled with melted paraffin and properly oriented to obtain mid-modiolar sections of the cochlea, and cross sections of the cristae ampullares, maculae utricle and saccule. Ten-micron-thick paraffin sections were obtained using a paraffin microtome (Leica, RM216S). The sections were then immediately mounted onto Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA, USA). The slides were dried on a slide warming plate for 30 min and stored at room temperature until use for IHC.

Deparaffinization

The slides with the tissue sections were placed in the oven at 65 °C for 10 min to remove paraffin. Slides were placed in a slide tray and immersed in 100 % xylene (5 × 3 min). Rehydration was performed by immersion with graded dilutions of ethanol (Fisher Scientific) for 5 min in each dilution as follows: 100 % three times, 70 % then 50 %. The slides were then placed in double distilled water and PBS for 10 min each.

Indirect IHC

Endogenous peroxidase inactivation in deparaffinized sections was performed by incubation for 10 min with 3 % hydrogen peroxide (diluted in 100 % in methanol). Slides were then washed with PBS for 10 min and then transferred to a Teflon slide carrier filled with antigen retrieval solution (Vector Antigen Unmasking Solution, Vector Labs, Burlingame, CA, USA) diluted 1:200 with double distilled water and heated in the microwave for a total of 5 min. The slides were cooled and transferred to a PBS wash for 10 min. Sections were incubated for 30 min with blocking solution containing 3 % normal rabbit or horse serum and 0.5 % Triton X-100 (Sigma) in PBS. Incubation with the respective primary antibody (Table 3) was performed for 48 h at 4 °C in a humid chamber. The sections were washed with PBS (3 × 5 min). Next, sections were incubated for 1 h with biotinylated secondary antibody, goat anti-rabbit polyclonal IgG (1:1000, Vector Labs, Burlingame, CA, USA). Afterward, sections were washed PBS (10 min × 3). Next, 1-h incubation was performed with Vectastain Elite ABC reagent (Vector Labs) followed by PBS washes (10 min × 3). Immunoperoxidase staining was performed using ImmPACT^® diaminobenzidine (DAB) solution (Vector Labs). The immunoreaction was stopped with PBS washes (10 min × 3). Slides were mounted with VectaMount AQ aqueous mounting media (Vector Labs) and glass cover slips.

IHC in celloidin-embedded sections

The methodology to mount celloidin-embedded sections, celloidin removal and antigen retrieval steps has been described in detail (O’Malley et al. 2009a, b; Shi et al. 1998) and used as described by our laboratory for the processing of human archival temporal bones (Ahmed et al. 2013; Balaker et al. 2013; Nguyen et al. 2014; Vorasubin et al. 2016). In brief, sections were removed from the archival jar and immediately floated 80 % ethanol and mounted on subbed glass slides (Superfrost Plus glass slides, Thermo Scientific). Bibulous paper (soaked with 10 % formalin) was placed over the section. A small roller was used to flatten the sections. A block of wood was placed over each slide. Sections were allowed to dry for 1 day, and the weight and the bibulous paper were removed.

Celloidin removal

A stock solution of sodium ethoxide was diluted with ethanol 1:3 (50 mL of this mixture is used per section). Celloidin sections previously mounted on Superfrost plus glass slides (O’Malley et al. 2009a) were placed in a glass Petri dish and immersed in the diluted solution for 30 min. Thereafter, sections were sequentially immersed in: 100 % acetone (30 min), 100 % methanol, 70, 50 % and distilled water (5 min each) and then immersed in hydrogen peroxide 5 % in methanol (10 min). Slides were then washed with distilled water before the antigen retrieval step.

Antigen retrieval

Glass slides with the tissue sections attached were placed horizontally in a glass Petri dish that contains antigen retrieval solution (Vector Antigen Unmasking Solution, Vector Labs, Burlingame, CA, USA), prepared at the time of use, diluted 1:200 with distilled water. Sections were heated in the microwave oven using intermittent heating methods of two 2-min cycles with and interval of 2 min between the heating cycles. The Petri dish containing the slides was removed from the microwave oven and allowed to cool for 15 min (room temperature) and washed with PBS for 10 min prior to IHC.

Indirect IHC

After antigen retrieval treatment, the celloidin sections were immunoreacted using the same protocol described above for paraffin-embedded tissue sections. Primary antibodies were incubated for 48 h at room temperature (Table 3). At the end of the IHC, the sections are covered with a water-soluble mounting media (Aqua-polymount, Polysciences) and let them dry for 1 day and imaged.

Indirect immunofluorescence in celloidin-embedded sections

Quenching of auto-fluorescence for immunofluorescence staining

After the antigen retrieval step, the sections were placed in a glass Petri dish containing iced cold PBS and placed in a UV chamber for 8 h. Temperature was checked constantly to avoid heating, and cold PBS was replaced every 30 min. This quenching step was mandatory to remove auto-fluorescence intrinsic to the human temporal bone sections. The presence of auto-florescence was checked before and after UV exposure. Sections were processed for IF once the auto-fluorescence signal in the tissue sections has disappeared.

Immunofluorescence

After auto-florescence was completely removed, the celloidin sections were immunostained using the same protocol described above for IF in formalin-fixed cryostat sections of the inner ear. The incubation step with hydrogen peroxide was omitted in tissue sections used for IF. Primary antibodies against prestin and acetylated tubulin (Table 3) were incubated for 48 h at room temperature.

Microscopic observation and documentation

Immunoreacted tissue sections were viewed and captured using an Olympus BX51 fluorescent microscope (Olympus America Inc., NY, USA) equipped with an Olympus DP70 digital camera. All images were captured using the same camera settings. Images were acquired using MicroSuite™ Five software (Olympus America Inc.). All images were prepared using the Adobe Photoshop™ software program run in a Dell Precision 380 computer. Confocal microscope images were acquired with a Carl Zeiss Laser scanning microscope model LSM 510 Meta Zeiss (Jena, Germany).