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Representative images of epidermal sheets are displayed in Figure 2B.
Immunohistochemistry of epidermal sheets and skin sections showed that LCs in VLP treated skin lost their typical dendritic morphology.
Indeed, direct en face observation of epidermal sheets at these time points revealed that H1 VLP treated samples contained high proportions of LCs with retracted dendrites, thus displaying a "rounded" morphology.
In addition, immunohistochemical analysis of epidermal sheets revealed that keratinocyte-restricted p65 deficiency did not affect the numbers of Langerhans cells in the epidermis (Supplementary Fig S1).
As a reliable indicator of in vivo senescence, DNA damage foci defined by phosphorylated H2A.X (γ-H2A.X) was increased in the whole mount of epidermal sheets including SG from p53 TSD /− mice.
It was demonstrated earlier that the whole-mount labeling of epidermal sheets can be used to visualize expression of stem cell markers [ 27], to trace label-retaining cells [ 17] and sebocytes [ 28], and so forth.
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Detection and visualization of CD207 was as for epidermal sheets.
Whole mount lipid staining of fixed epidermal sheets were stained with Nile Red (100 ng/ml in PBS) for 10 min, washed with PBS, mounted with VECTASHIELD containing DAPI, and immunofluorescence of the epidermal sheet was performed as previously described.
DC involves dorsalward extension of the lateral epidermal sheets and coordinated contraction of the AS cells to close the discontinuity and fuse the two epidermal flanks, thus forming a continuous epidermis.
Separation of dermal and epidermal sheets is based on a previously described procedure [38].
Following ID injection of influenza H1 VLP vaccine, a more pronounced effect on the number of LCs in epidermal sheets was observed.
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