Goat anti rabbit igg antibody conjugated with alexa fluor 488

The brain tissues were fixed in 4% paraformaldehyde at 4 °C overnight and immersed in 30% phosphate-buffered sucrose for 24 h, and then blocked in the Tissue-Tek OCT compound at −80 °C. Immunofluorescence was performed on 10 μm thick coronal sections. After being washed three times in PBS and permeabilized in 0.1% Triton X-100 for 10 min, sections were blocked in 2% fetal bovine serum (FBS) for 2 h at 37 °C and then incubated at 4 °C overnight with the following primary antibodies: mouse monoclonal anti-CypA antibody (1:50, Invitrogen) and rabbit monoclonal anti-PDGFRβ antibody (1:200, CST). Sections were rinsed in PBS and then incubated with the goat anti-mouse IgG antibody conjugated with Alexa Fluor 568 (1:200, Abcam), and goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:200, Abcam) for 1 h at 37 °C in the darkness. Finally, immunostaining images were observed under a fluorescence microscope (Olympus BX53, Olympus, Japan).

Briefly, microwave antigen retrieval was implemented in sodium citrate buffer (10 mM sodium citrate) at 95 to 100 °C for 10 min. Sections were blocked with 5% bovine serum albumin in phosphate-buffered saline (PBS) containing 0.3% Triton-X 100 for 1 h at RT. After washing with PBS, the brain slices were then incubated overnight at 4 °C with primary antibodies, including anti-HCN2 (1/200, Invitrogen, America), anti-Neun (1/200, Abcam, United Kingdom), anti-GFAP (1/200, Abcam, United Kingdom), and anti-iba1 (1/200, Abcam, United Kingdom). Two antibodies were co-incubated with sections to achieve bimolecular staining. Afterward, the brain slices were incubated with goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1/1000, Abcam, United Kingdom) or goat anti-mouse IgG antibody conjugated with Alexa Fluor 594 (1/1000, Abcam, United Kingdom) for one hour at RT in a dark room. Brain slices were mounted with Mounting Medium With DAPI—Aqueous, Fluoroshield (Abcam, United Kingdom) and then imaged under a fluorescence microscope. Immunostaining was analyzed by using the Image J software.

Vero-E6 cells (Cod. CRL-1586^™, ATCC®, USA), which were previously cultured in DMEM/F12 (HyClone, USA) + 10% fetal bovine serum (FBS) (HyClone, USA), were harvested and washed with DPBS with 5% FBS (FACS buffer). Approximately 10⁶ cells were blocked with FACS buffer and 5% of normal mouse serum (Abcam, USA) for 30 min at 37°C. Then, the cells were incubated with the purified RBD (8 μg/mL) for 2 h at 37°C. To remove the excess of RBD not attached to Vero E6, the cells were washed with FACS buffer twice. After that, the mix was marked with rabbit monoclonal antibody anti-SARS-CoV-2 S1 (1:200) (Sino Biological, China) as the primary antibody for 1 h at 37°C, followed by the addition of the secondary goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:200) (Abcam, USA). Finally, cells were acquired by the BD FACSCanto^™ II flow cytometer (BD Biosciences, USA). The data was analyzed using the software FlowJo v.10.6 (BD Biosciences, USA), and the graphics were generated with GraphPad Prism 8.0.1. For the interpretation of results, the percentage of positive cells indicates the binding of RBD to Vero E6 cells.