Histoembedder

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.

Chick eyeballs from the appropriate developmental stages were removed. Forty samples were used for the histology (10 samples per stage). The eyeballs were pricked with a needle so that the fixative could reach the inside. The cells were then fixed in 4% paraformaldehyde (P6148-500G, Sigma-Aldrich, St. Louis, MO, USA) and prepared with 2% sucrose in 0.1% M phosphate buffer (pH 7.4 needed to be maintained) for 24 h at 4 °C in the refrigerator. The next day, they were washed in PBS three times and dehydrated in an increasing percentage of alcohol starting from 70%, 80%, and 90% for an hour each, to 100% for 3 h with two changes. The eyeballs were then cleared in xylene (131769, Panreac, Barcelona, Spain) for 2 h and filtered through paraffin wax (P3558, Sigma-Aldrich). A tissue processor (Citadel 2000, Shandon, CA, USA) was used for the above processing. The filtered tissues were embedded in paraffin blocks using a Histo-Embedder (38621438, Leica, Wetzlar, Germany). The blocks were cut, and vertical sections were made at 4–5 μm thickness using a Rotary Microtome (Leica RM2235), stained with Meyer’s hematoxylin (MHS16, Sigma-Aldrich) and eosin (102439, Sigma-Aldrich), and mounted with DPX (10197905000, Merck, Darmstadt, Germany).

Following anesthesia, sterile 2/0 cotton ligatures (Polycot ®, Johnson e Johnson, São Paulo, SP, Brazil) were placed around the cervix of the right first inferior molar. The rats were immobilized at a custom made surgical table, allowing a reasonable mouth opening to place the cotton cord. The first right inferior molar was assigned to receive a ligature in a cervical position. The thread was introduced in the proximal space between the first and second molar with two small hemostatic clamps to hold the cotton cord. Two knots were made on the mesial face of the first molar and the ligatures were kept in position in order to allow plaque accumulation and pocket formation over a period of 27 days, according to Semenoff, Semenoff, Borges, Pedro, and Sakai (2010).
Periodontitis induction was confirmed by histology. Briefly, the tooth and surrounding soft tissue were removed in block and fixed in 10% formalin, decalcified for 45 days in Morse solution prepared by mixing equal volumes of 20% sodium of citrate (w/v) and 50% formic acid (v/v). Following dehydration (alcohol 70%—1 hr, 90%—1 hr, and 100%—16 hr) and paraffin embedding (Histoembedder ® Leica), 6 μm thick longitudinal sections in the mesiodistal direction were obtained and stained with hematoxylin and eosin (H/E) technique. The sections were examined histologically under a light microscope.