Jung rm2055

Cochlear histology was assessed in four bats. The bats were euthanized and perfused with PBSx1. The inner ears were dissected, fixed in 4% PFA at 4°C overnight, and decalcified at 4°C for 8 d in a standard decalcifier. The ears were then processed for paraffin sections using the tissue processor (TP1020; Leica). Paraffin blocks were then made using Histo-Embedder (Leica) and sectioned using a microtome (Jung RM2055; Leica). Cochlea mid-turn cuts were taken in all the samples for consistency. Paraffin serial sections (15 μm) were stained with hematoxylin and eosin using Multistainer (Leica). Slides were imaged using Aperio Slide Scanner (Leica). For quantification of the stria vascularis cross-sectional area, three slides were measured for each ear and averaged using FIJI (ImageJ2). Spiral ganglion neurons and the hair cells of an aged bat (11.3 yr) are shown in Fig S3. Both cell types seem intact, however, because of technical difficulties, we could not reliably quantify these cells’ survival.

Explants (5 × 5 mm) from the abdomen and vagina were immersed in fixative solution (4% paraformaldehyde) and embedded in paraffin with an automatic tissue processer (Thermo Shandon, Citadel 2000). Paraffin blocks were cut into 5 μm-thick serial sections with a microtome (Leica, JUNG RM2055) and placed on poly-L-lysine coated glass slides. Histological slides were observed using a binocular transmitted light microscope (Nikon Eclipse 80i) at 8X, 40X and 100X magnifications. Images were acquired using a digital camera (R3 Retiga, Qimaging) and processed with the software Image-Pro 10 (Media Cybernetics). Morphological, histological and immunohistochemical properties were analyzed.
The following staining was performed: hematoxylin–eosin (H&E) to evaluate the structure and grade of inflammatory reaction, Masson's Trichrome to evaluate the collagen fiber organization, and Sirius Red to evaluate the organization of collagen fibers (type I and III) of the explants. A macroscopic image at 100X magnification of each explant was performed (n = 10 hADM; n = 9 PP) and thereafter the images were quantified using ImageJ software (Launcher). The percentage of fibrosis was calculated as the stained area (SA) with respect to the total area (TA): (SA/TA)*100.

Eyes were embedded in paraffin and sections (8μm) were cut on a rotary microtome (Jung RM 2055, Leica, Nussloch, Germany). Antibodies against ERCC2 (Abcam plc, Cambridge, UK; order no ab102682; dilution 1:375) and γH2AX (Cell Signaling Technology, New England Biolabs GmbH, Frankfurt am Main, Germany; order no 9718, dilution 1:400) were used; binding to the corresponding antigen was visualized by a secondary antibody (Alexa Fluor488; Life Technologies GmbH, Darmstadt, Germany; dilution 1:250). Counterstaining of the cell nuclei was performed by DAPI (Sigma Aldrich Chemie GmbH, Taufkirchen, Germany; dilution 1:10.000). Stained sections were evaluated by a laser-scanning microscope (Olympus 1X81), equipped with imaging software Fluoview FV100 (Olympus Deutschland GmbH, Hamburg, Germany) and imported into an image-processing program (GIMP 2.8 or Photoshop CS6, Adobe Systems, Unterschleissheim, Germany).