This study was approved by our Institutional Animal Care Committee. Fifteen CBA/CaJ mice ranging in age from 4 weeks to 12 weeks were deeply anesthetized and intracardially perfused with one of 3 fixatives, 4% formaldehyde (F), 4% formaldehyde + 1% acetic acid (FA), and 4% formaldehyde + 1% acetic acid + 0.1% glutaraldehyde (FGA). The temporal bones were removed and fixed for an additional 25 hours at room temperature. Decalcification was accomplished using 120 mM ethylenediaminetetraacetic acid at a pH of 7 for seven days. The temporal bones were rinsed in distilled water and dehydrated in ethanols. In nine of the fifteen mice, the left ears were embedded in polyester wax (Electron Microscopy Sciences, Fort Washington, PA) and the right ears were embedded in celloidin (parlodion strips) (Mallinckrodt Chemicals, Phillipsburg, NJ). Both ears of the remaining six mice were embedded in paraffin (Paraplast X-TRA) (McCormick Scientific, St. Louis, MO). Temporal bones were sectioned at a thickness of 8-20 μm depending on the embedding medium. Paraffin sections were sectioned at 8 μm thickness and mounted on superfrost plus slides. Polyester wax sections were sectioned at 10 μm and mounted on superfrost slides coated with 0.5% bovine albumin and 0.5% fish gelatin [Merchant et al., 2006 (link)]. Celloidin sections were sectioned at 20 μm and those being immunostained were mounted on subbed glass slides smeared with albumin and fixed in place with formalin-soaked bibulous paper. A wooden block was placed on top of the bibulous paper and a 500g weight was placed on the block. The sections were dried for 1 hour under the weight.
Every tenth section was stained with hematoxylin and eosin and examined by light microscopy. Preservation of morphology within the scala media of the cochlea was assessed for each of the three fixatives and each of the three embedding media.
Prior to immunostaining, selected paraffin sections were dewaxed using xylenes, polyester wax was removed using ethyl alcohols [Merchant et al., 2006 (link)], and celloidin was removed using a solution of sodium hydroxide mixed in methanol [Miguel-Hidalgo and Rajkowska, 1999 (link)]. The sodium hydroxide methanol (NaOH-methanol) solution was made in the same manner as that described by Miguel-Hidalgo and Rajkowska; however the subsequent steps were modified. Once mixed, the solution was diluted 1:2 with methanol instead of 1:3 and used immediately. The amount of time the sections were exposed to the NaOH-Methanol solution was reduced from 30 minutes to 15 minutes (3 × 5 minutes) with 100% methanol rinses between each 5 minute change. The 3% H2O2step described by Miguel-Hidalgo and Rajkowska was omitted. The sections were hydrated from 100% methanol (10 minutes), through 70% methanol (10 minutes), and into distilled water. Following hydration, the sections were rinsed in 0.01 M phosphate buffered saline (PBS, pH 7.3), incubated in 5% normal horse serum for one hour, then incubated in primary antibodies overnight at room temperature in a humid chamber.
Immunostaining was accomplished using antibodies to prostaglandin d synthase (PGDS, Caymen) at a dilution of 1:5000, aquaporin 1 (Aqp1, Chemicon) at a dilution of 1:1000, connective tissue growth factor (CTGF, Cell Sciences) at a dilution of 1:10,000, 200 kD neurofilament (NF, Boehringer Mannheim) at a dilution of 1:2000, tubulin (Sigma) at a dilution of 1:15,000, and Na+,K+-ATPase (Siegel) at a dilution of 1:10,000. Following 14 hour primary antibody incubations, sections were rinsed in three washes of PBS. Secondary antibodies appropriate for the host species of primaries, at a dilution of 1:200, were applied and incubated for 1 hour. Following another three rinses in PBS, avidin-biotin-horseradish peroxidase (Standard ABC Kit, Vector Labs, Burlingame, CA) was applied to the sections and left to incubate for 1 hour, followed again by three washes with PBS. Finally, the sections were colorized using 0.01% diaminobenzidine and 0.01% H2O2 for between 5 and 10 minutes, rinsed, dehydrated, and cover slips were applied. Immunostaining was repeated at least once with each antibody, and in most cases, multiple times. The immunostained sections were analyzed by light microscopy and given a rating of no staining (−), adequate staining (+), good staining (+ +), or very good staining (+ + +).
Every tenth section was stained with hematoxylin and eosin and examined by light microscopy. Preservation of morphology within the scala media of the cochlea was assessed for each of the three fixatives and each of the three embedding media.
Prior to immunostaining, selected paraffin sections were dewaxed using xylenes, polyester wax was removed using ethyl alcohols [Merchant et al., 2006 (link)], and celloidin was removed using a solution of sodium hydroxide mixed in methanol [Miguel-Hidalgo and Rajkowska, 1999 (link)]. The sodium hydroxide methanol (NaOH-methanol) solution was made in the same manner as that described by Miguel-Hidalgo and Rajkowska; however the subsequent steps were modified. Once mixed, the solution was diluted 1:2 with methanol instead of 1:3 and used immediately. The amount of time the sections were exposed to the NaOH-Methanol solution was reduced from 30 minutes to 15 minutes (3 × 5 minutes) with 100% methanol rinses between each 5 minute change. The 3% H2O2step described by Miguel-Hidalgo and Rajkowska was omitted. The sections were hydrated from 100% methanol (10 minutes), through 70% methanol (10 minutes), and into distilled water. Following hydration, the sections were rinsed in 0.01 M phosphate buffered saline (PBS, pH 7.3), incubated in 5% normal horse serum for one hour, then incubated in primary antibodies overnight at room temperature in a humid chamber.
Immunostaining was accomplished using antibodies to prostaglandin d synthase (PGDS, Caymen) at a dilution of 1:5000, aquaporin 1 (Aqp1, Chemicon) at a dilution of 1:1000, connective tissue growth factor (CTGF, Cell Sciences) at a dilution of 1:10,000, 200 kD neurofilament (NF, Boehringer Mannheim) at a dilution of 1:2000, tubulin (Sigma) at a dilution of 1:15,000, and Na+,K+-ATPase (Siegel) at a dilution of 1:10,000. Following 14 hour primary antibody incubations, sections were rinsed in three washes of PBS. Secondary antibodies appropriate for the host species of primaries, at a dilution of 1:200, were applied and incubated for 1 hour. Following another three rinses in PBS, avidin-biotin-horseradish peroxidase (Standard ABC Kit, Vector Labs, Burlingame, CA) was applied to the sections and left to incubate for 1 hour, followed again by three washes with PBS. Finally, the sections were colorized using 0.01% diaminobenzidine and 0.01% H2O2 for between 5 and 10 minutes, rinsed, dehydrated, and cover slips were applied. Immunostaining was repeated at least once with each antibody, and in most cases, multiple times. The immunostained sections were analyzed by light microscopy and given a rating of no staining (−), adequate staining (+), good staining (+ +), or very good staining (+ + +).
