Proteomic analysis of formalin-fixed celloidin-embedded whole cochlear and laser microdissected spiral ganglion tissues

Markaryan, Adam; Nelson, Erik G.; Helseth, Lawrence D; Hinojosa, Raúl

doi:10.3109/00016481003591749

Cited by 6 publications

(6 citation statements)

References 16 publications

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“…Most of the human temporal bone banks have a collection of hematoxylin and eosin sections, from which there are remaining 9 out of 10 sections unstained that can be used for different types of staining, including IHC, histochemistry, molecular biology or proteomics (Jokay et al 1998; Kammen-Jolly et al 2001; Khetarpal 2000; Robertson et al 2001, 2006; Merchant et al 2008; Aarnisalo et al 2010; Markaryan et al 2008a, b, c, 2009a, b, c, 2010a, b, 2011; Maekawa et al 2010; Nelson and Hinojosa 2014). The tectorial membrane and Reissner’s membrane are generally well preserved, which often is not the case in paraffin-embedded or microdissected specimens.…”

Section: Discussionmentioning

confidence: 99%

“…Earlier studies utilized cytologic descriptions and graphic reconstructions of the cochlea (Guild 1921). Temporal bone science has advanced such that we are now entering a phase of methodological integration, whereby the same temporal bone can be used for light microscopy, transmission electron microscopy (TEM), immunohistochemistry (IHC), non-radioactive in situ hybridization, DNA and proteomics analysis (Aarnisalo et al 2010; Kong et al 1998; Merchant et al 2008; Ishiyama et al 2009, 2010; Lopez et al 2005a, b, 2007; Markaryan et al 2008a, b, c, 2009a, b, c, 2010a, b; Nguyen et al 2014; Richard et al 2015; Schrott-Fischer et al 1994, 2002a, b, 2007; Wackym et al 1990). The study of the human inner ear has lagged behind other areas of pathology, in large part due to the inaccessibility of the membranous labyrinth.…”

Section: Introductionmentioning

confidence: 99%

“…Thick celloidin-embedded sections (20–30 μm) of single temporal bone are kept immersed in 80 % ethylic alcohol for subsequent histological and/or immunohistochemical analysis including proteomics (Tian et al 1999; Aarnisalo et al 2010; Markaryan et al 2010a, b). Cytologic quantification of the cochlea is feasible when postmortem time is sufficiently short (Schuknecht 1993).…”

Section: Introductionmentioning

confidence: 99%

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Immunohistochemical techniques for the human inner ear

López

Ishiyama

Hosokawa

et al. 2016

Histochem Cell Biol

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Immunohistochemical techniques for the human inner ear

López

Ishiyama

Hosokawa

et al. 2016

Histochem Cell Biol

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“…Crockett et al used a FFPE cell-block of human lymphoma cell line. Recently, Markaryan et al (2010) used the Liquid Tissue Kit method to extract proteins from FFCE human cochlear tissue sections and spiral ganglion tissue isolated by laser microdissection. Protein identification was performed by bioinformatic analysis of high resolution tandem mass spectrometric data from fractionated tryptic peptide samples.…”

Section: Discussionmentioning

confidence: 99%

“…In the present study we detected 20 proteins from the cochlea identified by two or more peptides. Similarly, Markaryan et al (2010) identified 26 proteins with a minimum of two peptides. The present study and the study by Markaryan et al were both done using FFCE tissue.…”

Section: Discussionmentioning

confidence: 99%

A method for MSE differential proteomic analysis of archival formalin-fixed celloidin-embedded human inner ear tissue

et al. 2010

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Proteomic analysis of cadaveric formalin-fixed, celloidin-embedded (FFCE) temporal bone tissue has the potential to provide new insights into inner ear disorders. We have developed a liquid chromatography-mass spectrometry (LC-MS) method for tissue sections embedded with celloidin. Q-TOF (Quadripole-time of flight mass spectrometry) MS E (mass spectrometry where E represents collision energy) and Identity E ™ were used in conjunction with nano-UPLC (capillary ultrahigh pressure liquid chromatography) for robust identification and quantification of a large number of proteins.Formalin fixed paraffin-embedded (FFPE) mouse liver sections were used to evaluate formalin decross-linking by five different methods. Unfixed fresh mouse liver tissue was used as a control. Five different methods for preparation of FFPE tissue for MS analysis were compared, as well as four methods for celloidin removal with FFCE mouse liver tissue. The methods judged best were applied to FFCE 20 μm sections of mouse inner ear samples, and FFCE 20 μm human inner ear and human otic capsule bone sections.Three of the five tissue extraction methods worked equally in detecting peptides and proteins from FFPE mouse liver tissue. The modified liquid tissue kit protocol was chosen for further studies. Four different celloidin removal methods were compared and the acetone removal method was chosen for further analysis. These two methods were applied to the analysis of FFCE inner ear and otic capsule sections. Proteins from all major cellular components were detected in the FFCE archival human temporal bone sections. This newly developed technique enables the use of FFCE tissues for proteomic studies.

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Modulatory Effects of Mild Carbon Monoxide Exposure in the Developing Mouse Cochlea

2016

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Carbon monoxide (CO) is well known as a highly toxic poison at high concentrations, yet in physiologic amounts it is an endogenous biological messenger in organs such as the internal ear and brain. In this study we tested the hypothesis that chronic very mild CO exposure at concentrations 25-ppm increases the expression of oxidative stress protecting enzymes within the cellular milieu of the developing inner ear (cochlea) of the normal CD-1 mouse. In addition we tested also the hypothesis that CO can decrease the pre-existing condition of oxidative stress in the mouse model for the human medical condition systemic lupus erythematosus by increasing two protective enzymes heme-oxygenase-1 (HO-1), and superoxide dismutase-2 (SOD-2). CD-1 and MRL/lpr mice were exposed to mild CO concentrations (25 ppm in air) from prenatal only and prenatal followed by early postnatal day 5 to postnatal day 20. The expression of cell markers specific for oxidative stress, and related neural/endothelial markers were investigated at the level of the gene products by immunohistochemistry, proteomics and mRNA expression (quantitative real time-PCR). We found that in the CD-1 and MRL/lpr mouse cochlea SOD-2 and HO-1 were upregulated. In this mouse model of autoimmune disease defense mechanism are attenuated, thus mild CO exposure is beneficial. Several genes (mRNA) and proteins detected by proteomics involved in cellular protection were upregulated in the CO exposed CD-1 mouse and the MRL/lpr mouse.

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Proteomic analysis of formalin-fixed celloidin-embedded whole cochlear and laser microdissected spiral ganglion tissues

Cited by 6 publications

References 16 publications

Immunohistochemical techniques for the human inner ear

Immunohistochemical techniques for the human inner ear

A method for MSE differential proteomic analysis of archival formalin-fixed celloidin-embedded human inner ear tissue

Modulatory Effects of Mild Carbon Monoxide Exposure in the Developing Mouse Cochlea

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