Ix83 fluorescent microscope

Epidermal sheets were prepared as previously described (Mohammed et al., 2016 (link)). Briefly, epidermal side of shaved defatted flank skin or splitted ear skin was affixed to slides with double-sided adhesive (3M, St. Paul, MN). Slides were incubated in 10 mM EDTA in PBS for 90 min at 37°C, followed by physical removal of the dermis. The epidermal sheets were fixed in 4% PFA at RT for 15 min. The eidermal sheets were blocked with PBS containing 0.1% tween-20, 2% BSA and 2% rat serum or rabbit serum for 1 hour at RT before staining 1hour with antibodies at RT in PBS containing 0.1% tween-20 and 0.5% BSA. The slides were mounted with ProLong™ Gold Antifade Mountant (Thermo). Images were captured on an IX83 fluorescent microscope (Olympus Tokyo, Japan) using a x10 objective; image analysis was performed using cellSens Dimension software (Olympus). For enumeration of cells, three images from distant sites within an epidermal sheet from a mouse were counted (total 3 mm²) manually or automatically in ImageJ64 after image processing by Adobe Photoshop (version 6) and the average number per mm² epidermis was calculated as representative of the epidermal sheet. Anti-CD45.2 (104), CD8α (53–6.7), Thy1.1 (OX-7), MHCII (M5/114.15.2), hNGFR (ME20.4), YFP (FM264G) were purchased from Biolegend.

Epidermal sheets were prepared as previously described [25 (link)]. Shaved defatted flank skin was affixed to slides with double-sided adhesive (3M, St. Paul, MN). Slides were incubated in 10 mM EDTA in PBS for 90 min at 37°C, followed by physical removal of the dermis. The epidermal sheets were fixed in 4% PFA at room temperature (RT) for 30 min. The epidermal sheets were blocked with PBS containing 0.1% tween-20, 2% BSA and 2% rat serum for 1 hours at RT before staining overnight with Alexa Fluor 488–conjugated anti–GFP, Alexa Fluor 594–conjugated anti–mouse I-A/I-E and Alexa Fluor 647– conjugated anti-TCR γ/δ antibody in PBS containing 0.1% tween-20 and 0.5% BSA. Images were captured on a IX83 fluorescent microscope (Olympus Tokyo, Japan) using a x10 objective; image analysis was performed using cell Sens Dimension software (Olympus). Both LC and DETC in epidermis were counted either by ImageJ software or manually in a blinded fashion.

3T3 fibro-blasts were seeded onto PNIPAAm/PCL fibers at 630 000 cells per cm² and allowed to grow for 4 days. Medium was changed every day. Prior to detachment, cell were stained with 5 μM calcein, AM (ThermoFisher) and nuclear stain Hoescht 33342 (ThermoFisher) for 30 minutes. Detachment was initiated by rinsing 5 times over 10-15 minutes with cold (approximately 4 °C) medium to dissolve and remove PNIPAAm. Cell sheets were rinsed from the CellCrown insert with additional medium and imaged on an Olympus IX83 fluorescent microscope.