X tra adhesive slides

PUM spheroids were removed from the ULA plates using a cut 200-µl pipette tip at various time points and fixed in 10% neutral buffered formalin for 15 minutes. Spheroids were then suspended in 2% agar before being processed using the Bayer Tissue-Tek VIP E300 tissue processor (Bayer AG, Leverkusen, Germany). Processed spheroids were subsequently embedded in paraffin blocks and sectioned at 4 µm onto X-tra adhesive slides (Leica Biosystems, Wetzlar, Germany), for immunohistochemical (IHC) staining.
IHC staining was performed as previously described¹⁸ (link) using the Dako Pre-Treatment Module and the Dako Envision FLEX kit (Agilent Technologies, Santa Clara, CA), according to the manufacturer's instructions. Details of antibodies, antigen retrieval, and concentrations are provided in Table 1. Positive staining was visualized with an AEC substrate kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Sections were counterstained with Mayer's hematoxylin (VWR, Leighton Buzzard, UK), dyed blue with Scott's tap water (Leica), and mounted using Aquatex aqueous mounting medium (Sigma-Aldrich). Slides were scanned using the Leica Aperio CS2 slide scanner at 20× magnification.

Mice were euthanized on day zero and three weeks following subretinal transplantation; the eyes were enucleated and fixed in 4% formaldehyde (Merck, Darmstadt, Germany) overnight. The eyes were washed in PBS and then incubated for cryoprotection with 30% sucrose in PBS overnight at 4°C. Fixed tissue was embedded in OCT Tissue Freezing Medium (Scigen Scientific, Gardena, CA, USA) and frozen on dry ice. Cross-sections (10 μm) were placed on X-tra adhesive slides (Leica Biosystems, Peterborough, UK) and stored at -20°C.

Samples for histopathological examination were placed in 10% neutral buffered formalin for 7 days, and processed routinely to paraffin wax. Sections were cut at 5–6 μm, stained with haematoxylin and eosin (HE) and examined microscopically.
For immunohistochemistry, formalin-fixed, paraffin-embedded sections of spleen and liver, cut at 4 μm, were mounted on positively charged X-tra Adhesive slides (Leica Biosystems, UK), deparaffinised and rehydrated. Immunohistochemical staining was achieved using a BOND-MAX Immunostainer (Leica Microsystems, UK) and a Novacastra Bond Intense R (Leica Biosystems) detection kit. A heat-induced epitope retrieval cycle with buffer ER1 R (Leica Biosystems) was performed for 10 minutes. Slides were incubated with rabbit serum (4%) (Abcam, Cambridge, UK) for 20 minutes followed by an avidin/biotin blocking stage (15 minutes each) (Abcam). Polyclonal antibody raised in sheep immunised against recombinant CCHFv nucleoprotein (kindly provided by Dr John Barr, University of Leeds, UK) was incubated with the tissue for 30 minutes, followed by a biotinylated rabbit anti-sheep polyclonal antibody (Abcam) at a dilution of 1∶500, for 10 minutes. Haematoxylin was used as the counterstain. Positive and negative control slides were included. Immunolabelled slides were evaluated using light microscopy.