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.

Characterizing Adult cochlear supporting cell transcriptional diversity using single-cell RNA-Seq: Validation in the adult mouse and translational implications for the adult human cochlea

“…Immunohistochemistry on human temporal bones was performed as previously described by Lopez and colleagues (Lopez et al 2016). Detailed procedures of temporal bone collection, fixation, decalcification and celloidin embedding were described by Merchant (Merchant 2010).…”

“…The methodology to mount celloidin-embedded sections, celloidin removal and antigen retrieval steps has been described in detail (O’Malley, Merchant, et al 2009; O’Malley, Burgess, et al 2009; S. R. Shi, Cote, and Taylor 1998; Huang 1975) and used previously by the authors (Lopez et al 2016). Briefly, sections were removed from the archival jar and immediately floated in 80% ethanol and mounted on subbed glass slides (Superfrost Plus glass slides, Thermo Scientific).…”

“…Celloidin removal was performed by immersing the sections in sodium ethoxide diluted in ethanol (1:3 ratio) for 30 minutes followed by sequential immersions in 100% acetone, methanol (100%, 70%, 50% for 5 minutes each), distilled water, 5% hydrogen peroxide (10 minutes) and then washed with distilled water prior to antigen retrieval. Antigen retrieval was performed as described previously (Lopez et al 2016). Briefly, sections were heated in a microwave oven using intermittent heating methods of two 2-minute cycles with an interval of 2 minutes between the heating cycles in 1:200 diluted antigen retrieval solution in water (Vector Antigen Unmasking Solution, Vector Labs, Burlingame, CA, USA).…”

Supporting cell survival after cochlear implant surgery

“…Detailed procedures of temporal bone collection, fixation, decalcification, and celloidin embedding were described by Merchant . The methodology to mount celloidin‐embedded sections, perform celloidin removal and antigen retrieval steps, and perform immunohistochemistry on human temporal bones has been described previously . Sections were incubated with primary antibodies for 48 hours at room temperature and secondary antibodies for 2 hours at room temperature.…”

In Focus in HCB

“…We will briefly highlight two of those articles here. Lopez et al (2016) provide a comprehensive review of methods and protocols for immunohistochemical studies of the human inner ear. Since the first decision often encountered in any prospective immunohistochemical study is choice of sample processing and potential embedding media, they compare and contrast immunostaining results obtained for inner ear tissue samples (as well as microdissected auditory and vestibular endorgans from human temporal bone) processed for subsequent frozen sections, paraffin sections and celloidin sections.…”