Fig 1: Impairment of Reticulon 1A (RTN1A) functionality instigates a roundish morphology in resident Langerhans cells (rLCs).(A) Experimental workflow demonstrating the cultivation of human epidermal sheets with an a-RTN1A antibody (ab) and the isotype for indicated time points and subsequent analysis strategies. (B–C) Representative immunofluorescence (IF) images of isotype- and a-RTN1A-treated epidermal sheets at indicated time points stained for CD207 (green), a secondary ab (red) to visualize the uptake of the isotype and a-RTN1A ab by rLCs and nuclear counterstaining with 4',6-diamidino-2-phenylindole (DAPI) (blue). Zoom-ins of the boxed areas are also shown as 3D projections underneath. RTN1A+ rLCs: yellow arrows, RTN1A- rLCs: white arrows. n=3, scale bar: 20 µm. (D) Representative IF images showing RTN1A, CD207, vimentin, and DAPI staining in untreated (UT), isotype- and a-RTN1A-treated human epidermal sheets after 24 hr of cultivation. Co-localization of RTN1A with vimentin: pinkish arrows, rLCs with partially retracted dendrites: white arrows. n=4, scale bar: 20 µm. (E, F) Enumeration, percentage, and distribution of dendritic and roundish rLCs in epidermal sheets upon 24 hr of culture and indicated treatment. (E) Data are shown as standard error of the mean (SEM) from four fields of view (FOVs) of four donors and were analyzed using two-way ANOVA with Tukey’s multiple-comparison test. (F) Data represent mean of four donors. *p=0.05, **p=0.01.
Fig 2: Hampering Reticulon 1A (RTN1A) function decreases the Langerhans cell (LC) migration potential and alters the marker expression in emigrated LCs (eLCs).(A, B) Enumeration and ab uptake of pre-gated CD207+CD1a+ eLCs, collected from culture wells with epidermal sheets at indicated time points and analyzed via flow cytometry. (A) Data are presented as standard error of the mean (SEM) from triplicates of three donors. Ordinary one-way ANOVA with Tukey’s multi-comparison test was used. *p=0.05. (B) Data from triplicates, including three donors, are shown as mean fold change to untreated (UT) eLCs. (C) Marker expression profile of pre-gated CD207+ eLCs upon 24 hr of culture, treatment, and flow cytometry analysis. Data shown represent mean ± SEM of three donors and were analyzed using two-way ANOVA Tukey’s multi-comparison test. *p=0.05. (D) Adhesion molecule concentrations in supernatants of cultivated epidermal sheets with indicated 24 hr treatments and subsequent LEGENDplex bead array measurement. Data are shown as SEM from duplicates of four donors and were analyzed using two-way ANOVA with Tukey’s multiple-comparison test. *p=0.05, **p=0.01.
Supplier Page from MilliporeSigma for Anti-CD207 antibody produced in rabbit