Epon 812

Muscle tissue was prepared as described in Schwarz et al. (2000) (link). For the preparation of rodent brain tissue the animals were perfused transcardially first with 30 ml of phosphate-buffered saline and then with 40 ml of fixative solution (4% paraformaldehyde in 0.1M PBS [pH 7.4]). The brain tissue was then removed and kept in fixative over night at 4 °C. After being washed twice in PBS, tissue slices (0.2 to 1.5 mm thick) were cut on a vibratome (752 M Vibroslice, Campden Instruments, Leichester, United Kingdom) and kept for 24 h in PBS at 4 °C. Pieces about 1.5 mm in size were then excised and washed three times for 30 min each in cacodylate buffer at pH 7.4.The tissue was postfixed for 2 h in 2% osmium tetroxide/1.5% potassium ferric cyanide in aqueous solution at room temperature. Then the tissue was subjected to a contrast enhancement step by soaking it over night in a solution of 4% uranyl acetate in a 25% methanol/75% water mixture (Stempak and Ward 1964 (link)) at room temperature. After that the tissue was dehydrated in a methanol sequence (25%, 70%, 90%, and 100% for 30 min each) followed by infiltration of the epoxy (Spurr, Epon 812, or Araldite, all from Serva, Heidelberg, Germany) monomer (epoxy/methanol 1:1, for 3 h rotation at room temperature; epoxy/methanol 3:1, overnight at 4°C; pure epoxy, 3 h rotating at room temperature). Polymerization was 48 h at 60 °C for Epon and at 70 °C for Spurr and Araldite. The block face was trimmed to a width of several hundred microns and a length of about 500 μm using either a conventional microtome or a sharp knife. SEM images of the untrimmed block face can be used to select the desired field of view before the final trimming step producing the desired small cutting pyramid.

After completing the ABR measurements at 1, 3, 6, and 9 month of age at The Jackson Laboratory the inner ears were dissected out, immersed in 4% paraformaldehyde and shipped to the University at Buffalo for analyses of the cochlea and vestibular system. Our procedures for preparing cochleograms showing the percentage of missing inner hair cells (IHC) and outer hair cells (OHC) as a function of percent distance from the apex have been described in detail previously [14 (link), 15 (link), 122 ]. Mice evaluated by ABR at the Jackson Lab were euthanized by CO₂ asphyxiation and decapitated. The temporal bones were removed, immersed in 4% paraformaldehyde, and shipped to the University at Buffalo for analysis. Cochleae were stained with Ehrlich's hematoxylin solution, the organ of Corti dissected out as a flat surface preparation, mounted in glycerin on glass slides and coverslipped. A person, blind to the results, dissected the cochleae and prepared the surface preparation. A second person blind to the experimental conditions counted the hair cells using a light microscope (Zeiss Standard, 400X magnification). By raising and lowering the focal plane, the investigator can determine if the hair cell nucleus, cuticular plate and stereocilia bundle were present. A hair cell was counted as present if both the cuticular plated and nucleus were clearly visible and considered missing if either were absent. OHC and IHC were counted along successive 0.12-0.24 mm intervals from the apex to the base. Using lab norms and custom software, the percentage of missing IHC and OHC were determined for each animal and a cochleogram was constructed showing the percentage of missing OHC and IHC as a function of percent distance from the apex of the cochlea. Position in the cochlea was related to frequency using a mouse tonotopic map [33 (link)]. In some cases, the cochlear surface preparations were photographed with a digital camera (SPOT Insight, Diagnostic Instruments Inc.) attached to a Zeiss Axioskop microscope, processed with imaging software (SPOT Software, version 4.6) and Adobe Photoshop 5.5.
To evaluate the condition of the cochlea and vestibular sensory epithelium in more detail, some inner ears were embedded in plastic using procedures described in our earlier publications [32 (link), 122 , 123 (link)]. Following fixation, inner ears were decalcified (Decal, Baxter Scientific Products), rinsed in phosphate buffered saline, dehydrated through a graded series of EtOH and then embedded in Epon 812 (Electron Microscopy Sciences). Sections were cut parallel to the modiolus of the cochlea at a thickness of three μm on an ultramicrotome, stained with 0.5% toluidine blue, mounted on glass slides, examined with a Zeiss microscope (Axioskop) and photographed with a digital camera as above. Sections (3 μm) were also taken from the utricle, saccule and crista ampullaris following similar procedures.

Cells were scraped from plates and collected by centrifugation. Samples were sequentially fixed in 3% glutaraldehyde and 1% osmium tetroxide, cooled on ice for 1 h, washed with 0.1 M cacodylate buffer (pH 7.2) containing 0.1% CaCl₂, and dehydrated in an ethanol and propylene oxide series. Next, samples were embedded in Epon 812 mixture and polymerized at 60°C for 36 h. Using the ULTRACUT UC7 ultramicrotome (Leica Biosystems, Vienna, Austria), sections of 70 nm thickness were cut and mounted on 75-mesh copper grids. Sections were counterstained with uranyl acetate and lead citrate for 10 min and 7 min, respectively, and examined using the KBSI Bio-High Voltage EM (JEM-1400 Plus at 120 kV and JEM-1000BEF at 1000 kV; JEOL Ltd., Tokyo, Japan).

After fixation, the tissue samples were rinsed in phosphate-buffered saline solution and postfixed in 1% osmium tetroxide, dehydrated through an ascending series of ethanol, immersed in propylene oxide, and embedded in Epon 812 (Agar Scientific, Essex, UK). Afterwards, ultrathin (60–90 nm) sections were obtained using a Leica EM UC7 ultramicrotome (Wetzlar, Germany) mounted on copper grids, contrasted with saturated uranyl acetate and lead citrate, and examined by transmission electron microscopy (TEM, Zeiss-EM 109 electron microscope, Germany). The appearance, organelles distribution, membranes integrity, and connections between the granulosa cells and the oocyte were investigated.