Collagen Fibril Diameter Analysis in Murine Spinal Discs

Lumbar spine discs (L4/5) and caudal spine discs (C7/8) from 4-week-old IFT80^fl/fl; Col1-creERT or IFT80^fl/fl mice were dissected and fixed in 2.5% (v/v) glutaraldehyde at 4 °C. The collagen fiber diameters were measured using SEM analysis as described previously (25 (link)). Briefly, the samples were digested in 20.4 U/ml hyaluronidase (H3506, Sigma) and 0.1 mg/ml bovine pancreatic trypsin (T1426, Sigma-Aldrich). The fixed samples were washed three times with PBS. The specimens were dehydrated in a graded ethyl alcohol (EtOH) series (30%, 50%, 70%, 80%, 90%, and 100%). Subsequently, specimens were dehydrated in ethanol and hexamethyldisilizane (HMDS) solutions, starting with EtOH: HMDS (1:1) and serially increasing to EtOH: HMDS (1:4), and finally washed with 100% HMDS. The samples were air dried in the fume hood for one hour. Samples were mounted on the AI-hold with super glue and coated with carbon. The FEI XL30 ESEM (FEI XL30 ESEM, FESEM Thermo Fisher, 5350 NE Dawson Creek Drive, Hillsboro, Oregon 97124 USA, voltage: 8 kV) was used for imaging. ImageJ software was used for the measurement of collagen fibril diameters. Six mice were evaluated in each group.