Eosin y

For the observation of B-cell follicles, mantle zones, germinal centers, and LN morphology, H&E/IgD “double” staining was performed. Slides of 2 µm were dried at 58 °C overnight and deparaffinized in xylene and decreasing ethanol concentrations. Heat-mediated epitope retrieval was performed for 20 minutes at pH 6.1 and 95 °C. Slides were stained with hematoxylin before further proceeding with IgD staining. IgD immunohistochemistry was performed on an automated staining system using a horseradish peroxidase catalyzed brown chromogen reaction according to the manufacturer’s guidelines (antibody: polyclonal rabbit anti-human IgD, IS51730-2, ready to use, Dako, Hamburg, Germany; staining: Autostainer Plus, Dako). The slide was then stained with Eosin Y (Leica Biosystems, Nussloch, Germany) and covered with a coverslip.

For routine histology, tissue was either fixed in 4% paraformaldehyde (30 min) and embedded in paraffin or embedded and frozen in optimal cutting temperature (OCT) medium. Five µm thick sections were cut and stained as described previously [24 (link)]. Briefly, sections were stained with hematoxylin (Haematoxylin Gill III, Surgipath, Leica, Germany) for 2 min and 1% eosin Y (Surgipath, Leica, Germany) for 1 min to observe the gross tissue architecture and degree of decellularization. To visualize the proteoglycan content, cryosections were stained with 1% alcian blue (Morphisto, Offenbach am Main, Germany) for 30 min together with a counterstain of nuclear fast red (Morhphisto, Offenbach am Main, Germany). For glycoproteins, PAS staining was performed using 1% periodic acid (Honeywell Fluka, Charlotte, NC, USA) for 10 min, and Schiff reagent (Roth, Karlsruhe, Germany) for 90 s. Samples were examined using either a Hamamatsu NanoZoomer S60 (Hamamatsu Photonics, Herrsching, Germany) or a bright field fluorescence microscope (Axio Imager.A1, Zeiss) and images were processed using ProgRes CapturePro Software (JENOPTIK, Dreseden, Germany)).

For bright field light microscopy, tissue was cryosectioned and stained as follows: After embedding and freezing in optimal cutting temperature medium, 10 µm thick sections were cut with a cryostat (Leica, Germany), mounted on adhesive slides and air-dried. Sections were stained with hematoxylin (Haematoxylin Gill III, Surgipath, Leica, Germany) for 2 min and 1% eosin Y (Surgipath, Leica, Germany) for 1 min to observe the gross tissue architecture and degree of decellularization. To visualize the proteoglycan content, cryosections were stained with 1% alcian blue (Morphisto, Germany) for 30 min together with a counterstain of nuclear fast red (Morhphisto, Germany). For glycoproteins, PAS staining was performed using 1% periodic acid (Fluka, Germany) for 10 min, and Schiff reagent (Roth, Germany) for 90 s. Samples were examined using either a bright field fluorescence microscope (Axio Imager.A1, Zeiss) and images were processed using ProgRes CapturePro Software (JENOPTIK, https://www.jenoptik.com/products/cameras-and-imaging-modules/microscope-cameras/progres-usb-20-firewire/software-download-progres) or a Hamamatsu NanoZoomer S60 (Hamamatsu Photonics, Herrsching, Germany).