Whole brains and whole mount sections were imaged using a Zeiss Axio Imager.Z1 and ApoTome I or a Cairn Custom designed dissection microscope. Brains were then sucrose-treated (15 and 30% sucrose solution sequentially for 24 h each), OCT-embedded, and then 20 μm thick sections were cut using a cryostat.
These sections were then stained in a solution containing 10% BSA and 0.1% triton, using the primary antibodies ACE-2 (1:50 R&D AF933), aquaporin-1 (1:100 Merck ab2219), CD3 (1:100 Bio-Rad MCA1477), CD20 (1:100 Abcam ab219329), CD68 (1:100 DAKO M0814), claudin-5 (1:100 Invitrogen 35-2500 (4C3C2)), cleaved caspase-3 (1:100 Cell Signalling Technology 9661), ERG (1:100 Abcam ab92513), Hopx (1:100 Santa Cruz sc-398703), HuC/D (1:100 Abcam ab184267), IBA-1 (1:100 Abcam ab5076), MAP2 (1:100 Millipore ab5622), Mouse IgG isotype control (1:100 Sigma M5284), nestin (1:100, Sigma N5413), pan-laminin (1:100 Sigma L9393), Rabbit IgG isotype control (1:50 Abcam ab172730 [EPR25A]), SARS-CoV-2 spike protein (1:100-250 Genentech GTX632604 [1A9]), SARS-CoV-2 nucleocapsid protein (1:50-150 Sino Biological 40143-R001), S100a9 (1:200 BMA biomedicals T-1026), Sox2 (1:100 R&D Systems AF2018).
The Life technologies secondary antibodies, used at 1:1000, were donkey-anti-goat Alexa fluor 488 (A11055), anti-mouse Alexa fluor 568 (A10037) and 555 (A31570), anti-rabbit Alexa fluor 647 (A31573) and 488 (A21206), and goat anti-rat 555 (A21434) and anti-mouse 594 (A11005). Sections were all stained with DAPI (Sigma D9542) and mounted in Mowiol (Merck Biosciences).
Sections were then imaged using either a Zeiss LSM 800 inverted confocal microscope and a Zeiss Plan-Apochromat 20 × 0.8 objective, or a Zeiss AxioScan slide scanner and a Zeiss Plan-Apochromat 20 × 0.8 M27 objective.
These sections were then stained in a solution containing 10% BSA and 0.1% triton, using the primary antibodies ACE-2 (1:50 R&D AF933), aquaporin-1 (1:100 Merck ab2219), CD3 (1:100 Bio-Rad MCA1477), CD20 (1:100 Abcam ab219329), CD68 (1:100 DAKO M0814), claudin-5 (1:100 Invitrogen 35-2500 (4C3C2)), cleaved caspase-3 (1:100 Cell Signalling Technology 9661), ERG (1:100 Abcam ab92513), Hopx (1:100 Santa Cruz sc-398703), HuC/D (1:100 Abcam ab184267), IBA-1 (1:100 Abcam ab5076), MAP2 (1:100 Millipore ab5622), Mouse IgG isotype control (1:100 Sigma M5284), nestin (1:100, Sigma N5413), pan-laminin (1:100 Sigma L9393), Rabbit IgG isotype control (1:50 Abcam ab172730 [EPR25A]), SARS-CoV-2 spike protein (1:100-250 Genentech GTX632604 [1A9]), SARS-CoV-2 nucleocapsid protein (1:50-150 Sino Biological 40143-R001), S100a9 (1:200 BMA biomedicals T-1026), Sox2 (1:100 R&D Systems AF2018).
The Life technologies secondary antibodies, used at 1:1000, were donkey-anti-goat Alexa fluor 488 (A11055), anti-mouse Alexa fluor 568 (A10037) and 555 (A31570), anti-rabbit Alexa fluor 647 (A31573) and 488 (A21206), and goat anti-rat 555 (A21434) and anti-mouse 594 (A11005). Sections were all stained with DAPI (Sigma D9542) and mounted in Mowiol (Merck Biosciences).
Sections were then imaged using either a Zeiss LSM 800 inverted confocal microscope and a Zeiss Plan-Apochromat 20 × 0.8 objective, or a Zeiss AxioScan slide scanner and a Zeiss Plan-Apochromat 20 × 0.8 M27 objective.
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