Ab179707

Euthanized animals were perfused with 12 ml of saline, after which brains were harvested, separated into ipsilateral and contralateral hemispheres, and snap frozen in liquid nitrogen. Samples were lysed in a 500 µl RIPA buffer containing protease and phosphatase inhibitors in combination with a Tissuelyser LT system (Qiagen), followed by centrifugation. Protein concentration was evaluated in the supernatants by the BCA assay (Pierce). Samples were then denatured with Laemmli sample buffer, and western blotting was conducted using TGX 4–20% gradient precast gels (BioRad) and a turboblot transfer system (BioRad). Proteins were targeted with the following antibodies: Iba1 (Abcam ab17884, 1:1000), GFAP (Cell Signaling 12389, 1:1000), ICAM1 (Abcam ab179707, 1:750), and GAPDH (Novus Biologicals NB600–502FR). Detection was performed with corresponding 800cw tagged antibodies (Licor 926–32213; 1:20000) and anti-GAPDH Dylight 680 (Novus Biologicals NB600–502FR), followed by scanning in the Licor CLx system (Licor). Quantification was performed using Image studio software (Licor).

Paraffin slides with 5 μm thick tissue sections were deparaffinized with xylene and ethanol. Antigen retrieval performed with sodium citrate buffer (Sigma-Aldrich C9999) and a steamer. Primary antibodies were incubated overnight at 4°C, and Alexa Fluor secondary antibodies were incubated at room temperature for 2 hours prior to tissue mounting with Prolong Gold (Thermo Fisher P36930). Importantly, all control and experimental samples were embedded on the same slide and underwent staining under identical conditions. Imaging was performed using identical settings.
Primary antibodies: pan-ACE2 (goat, 1:1,000, R&D AF933), hACE2 (rabbit, 1:200, Abcam ab108209), E-cadherin (rabbit, 1:200, Cell Signaling 3195S), E-cadherin (goat, 1:200, R&D AF748), olfactory marker protein (goat, 1:200, Wako 544–10001), SARS-CoV-2 nucleocapsid (rabbit, 1:500, Rockland 200-401-A50), PDPN (hamster, 1:500, Novus Biologicals AB15858), DC-LAMP (rat, 1:25, Novus Biologicals, DDX0191P-100), ICAM-1 (rabbit, 1:500, Abcam ab179707), vWF (rabbit 1:1,000, Novus Biologicals NB600-586), Endomucin (rat, 1:100, Abcam ab106100), β3-Tubulin (mouse, 1:1,000, Abcam ab78078), NeuN (mouse, 1:1,000, Novus Biologicals NBP1-92693), GFAP (chicken, 1:1,000, Novus Biologicals NBP1-05198), Krt8 (rat, 1:100, DSHB, TROMA-I).

Immunohistochemistry staining was performed as previously described (23 (link)) with control and experimental samples on the same slide and under identical staining conditions. Primary antibodies were as follows: pan-ACE2 (goat, 1:1,000, R&D AF933), hACE2 (rabbit, 1:200, Abcam ab108209), SARS-CoV-2 nucleocapsid (rabbit, 1:500, Rockland 200-401-A50), ICAM-1 (rabbit, 1:500, Abcam ab179707), vWF (rabbit 1:1,000, Novus Biologicals NB600-586), and PECAM (goat 1:500, R&D AF3628). Fluorescence-conjugated Alexa Fluor secondary antibodies were used (1:500, Invitrogen) according to the primary antibody species and counterstained with DAPI (1:1,000). ICAM1 and vWF fluorescence intensity were calculated by integrated fluorescence intensity. All images were analyzed using ImageJ/FIJI software.