Fig. 4

Apical and Basal TfR membrane domain localization in Brain Endothelial Cells using Expansion Microscopy. Normal immunofluorescence (IF) confocal micrographs (a, b, pre-expansion) served to control for sufficient isotropic expansion (c) and staining pattern of expansion specimens (d–k). Representative pre-expansion IF images show BECs established on collagen IV pre-coated filter membranes in non-contact co-culture (NCC) with astrocytes under normal and retended endoplasmic-Golgi trafficking conditions from Brefeldin A (BFA) treatment (c) with images presented as maximum intensity z-projections of nuclei (blue), collagen IV (red) and TfR (green) stainings, obtained by confocal microscopic imaging with 60 × magnification (UPlanSApo 60X, NA 1.20, water objective lens). Top view illustrates the rough overview of TfR distribution patterns while the below orthogonal view illustrates the limitation of a detailed overview using the normal IF technique. Expansion Microscopy (ExM) allowed visualization of the detailed TfR localization, with confocal micrographs of expanded BEC specimens in orthogonal view (e–g, i–k) showing nuclei staining (blue), collagen IV staining (red) marking the basal membrane localization, and TfR staining (green). Representative single slides present the capability of differentiating the apical and basal membranes and visualize TfR distribution, with white arrows marking basal TfR. Scale bar is equal to 10 μm for micrograph a–c and 1.6 μm for micrograph d–k