Mouse anti human involucrin sy5

Cornified envelopes were isolated from the tape using an established methodology [29 ]. Briefly, half of each tape was extracted with 750 mL of dissociation buffer containing 100 mM Tris-HCl pH 8.0, 5 mM EDTA (ethylenediaminetetraacetic acid), 2% SDS (sodium dodecyl sulphate) and 20 mM DL-dithiothreitol (Sigma Aldrich Dorset, UK). Tapes were extracted in the dissociation buffer for 10 min at 75 °C and centrifuged at room temperature for 10 min at 5000 g. The extracted CEs were washed (three times) in washing buffer: 20 mM Tris-HCl pH 9.0, 5 mM EDTA, 0.2% SDS and 10 mM DL-dithiothreitol and suspended in 1 $\times$ PBS buffer (Sigma Aldrich Dorset, UK).
Extracted CEs were transferred onto a Polysine-coated microscope slide (5 μL, VWR international Ltd, Leicestershire, UK) for the immunostaining protocol, as previously described [29 ]. This included an overnight incubation in a humidity chamber at 4 °C in the primary monoclonal antibody (1:100, mouse anti-human involucrin SY5, ABCAM, Cambridge, UK). The antibody solution was washed with PBS three times for 5 min before adding the secondary antibody Alexa-Fluor 488-labeled goat anti-mouse IgG antibody (1:200, ABCAM, Cambridge, UK) for 60 min at room temperature (in the dark). The slides were washed with 1 $\times$ PBS (three times for 5 min) and mounted with 20 μg/mL Nile red (Sigma Aldrich, Dorset, UK) in 75% glycerol solution.