Tissue preparation, immunohistochemistry, and microscopy

Tissue from hole-punched ears was fixed with Prefer fixative (the active ingredient is glyoxal) (Anatech) overnight, washed in H₂0, and placed in 70% ETOH. The tissue was embedded in paraffin and then cut into 5-μm-thick sections. Before staining, slides were dewaxed in xylene and rehydrated. Antigen retrieval was performed by autoclaving for 20 min in 10 mM sodium citrate, pH 6.0. The tissue sections were then treated with 0.1% Triton, and nonspecific binding was blocked with 4% BSA (A7906; Sigma) for 1 h. The primary antibodies and matched secondary antibodies used for immunohistochemistry (IHC) are shown in Table 1.

Primary and secondary antibodies used for IHC.

For histological stains, tissue sections were treated the same, as explained in the previous section, and then stained with hematoxylin (Leica Microsystems, # 3801562) and eosin (Leica Microsystems, #3801602). For IHC, tissue sections were then treated with 0.1% Triton, and nonspecific binding was blocked with 4% BSA (A7906; Sigma) for 1 h. The primary antibodies and matched secondary antibodies used for IHC are shown in Table 1. The slides were washed, rehydrated, cleared with xylene, and coverslipped with Permount mounting medium (Fisher, SP15-500). Staining was visualized for fluorescent labeling using a fluorescent Olympus (AX70) microscope and a DP74 camera and cellSens software for image analysis, or a bright-field microscope for H&E staining, as previously described (Zhang Y. et al., 2015).

For cultured cell staining, primary fibroblast-like cell lines from ear tissue were established from MRL and B6 female mice and then grown in DMEM–10% FBS supplemented with 2 mM L-glutamine and 100 IU/mL penicillin–streptomycin, and maintained at 37°C, 5% CO₂, and 21% O₂. For immunohistochemical staining, fibroblasts were grown on coverslips in DMEM with 10% FBS at 37°C in a humidified 5% CO₂ incubator. The coverslips were rinsed with 1× PBS; the cells were fixed in cold methanol (−20°C) for 10 min, rinsed with 1× PBS, treated with 0.1% Triton-X100, and then incubated with the appropriate primary and secondary antibodies (Table 1). Photomicrographs were produced using the fluorescent microscope (Olympus AX70) and a DP74 camera, with cellSens Standard software for image analysis.

Confocal images were captured using a Leica TCS SP5 II laser scanning confocal microscope (Leica Microsystems, Inc., Deerfield, IL) using AOBS and sequential scanning with 405, 488, and 561-nm laser lines. Individual frames and short z-stacks were acquired at maximum resolution with a 63 × 1.4 NA objective, following Nyquist criteria. Post-processing for maximum projection and noise reduction was carried out using Leica LAS-AF software and exported to .tif files.