Alexa fluor 488 conjugated goat anti rabbit igg h l

The rats were anesthetized and perfused with 4% paraformaldehyde, then the brain tissues were collected, fixed and sliced. Next, 10% normal donkey or goat blocking serum (Solarbio, Beijing, China) was used to block brain tissues sections for 30 min at 37°C. The sections (10 μm) were then incubated at 4°C overnight with the following primary antibodies: TSPO (1:100 dilution, pa5-19088; ThermoFish Scientific), Iba1 (1:200 dilution, gb12105; Servicebio), ATG7 (1:100 dilution, ab133528; Abcam), LC3B (1:100 dilution, ab192890; Abcam) and p62 (1:100 dilution, ab109012; Abcam). The secondary antibodies Cy3 conjugated Donkey Anti-Mouse IgG (H+L) (1:200 dilution, gb21401; Servicebio), FITC conjugated Donkey Anti-Goat IgG (H+L) (1:200 dilution, gb22404; Servicebio), Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG (H+L) (1:400 dilution, gb25303; Servicebio) and Cy3 conjugated Goat Anti-mouse IgG (H+L) (1:300 dilution, gb21301; Servicebio) were used and incubated 1 h in the dark. The sections were incubated with Hoechst (1:600 dilution, g1011; Servicebio) for 3 min for nuclear staining. The sections were analyzed and photographed using a laser scanning confocal microscope (NIKON Eclipse Ti, Japan). Finally, Image J performs quantitative analysis.

Sections were routinely dewaxed and dehydrated. The sections were permeated with 0.5% Triton X-100 after repair of antigen, then incubated with 10% goat serum for 1 h. The samples were incubated with primary antibodies, rat anti-iNOS (1:200, Servicebio, China), and mice anti-Iba-1 (1:200, Servicebio, China) at 4 °C overnight. The samples were also incubated with Alexa Fluor 488 conjugated Goat Anti-rabbit IgG (H⁺L) (1:500, Servicebio, China) and Cy3 conjugated Goat Anti-mouse IgG (H⁺L) (1:300, Servicebio, China) secondary antibodies for 1 h. The sections were also incubated with DAPI for 5 min and visualized via fluorescence microscopy (Leica DM IL LED microscope, Wetzlar, Germany). Eight randomly selected fields were used to calculate the average fluorescence intensity of iNOS and Iba-1 via ImageJ 1.5.1 software.

Cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. Paraformaldehyde-fixed, paraffin-embedded tissue samples was cut into 4 μm slices and adhered to frosted glass slides. After washing with PBS and treating with PBS containing 0.1% Triton X-100 for 15 min, the sections and cells were permeabilized and blocked with 0.1% Tween-20 in PBS (PBST) containing 5% FBS for 90 min at room temperature. The cells were immunostained with anti-ACE2 (ab15348, 1:500, Abcam), anti-SP1 (T453) (ab59257, 1:500, Abcam), or anti-HNF4α antibodies (3113, 1:1 000, Cell Signaling Technology) overnight at 4 °C. The tissue sections were immunostained with anti-ACE2 (GB11267, 1:200, Servicebio, Wuhan, China) or anti-SARS-CoV-2-N antibodies (40143-MM05, 1:500, SinoBiological, Beijing, China) overnight at 4 °C. After washing three times with 0.1% Tween-20 in PBS (PBST), the cells were incubated with Alexa Fluor 594 anti-rabbit IgG (H+L) (A-21207, 1:200, ThermoFisher Scientific), Cy3 conjugated goat anti-mouse IgG (H+L) (GB21301, 1:300, Servicebio), or Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) (GB25303, 1:500, Servicebio) at room temperature for 1 hr. After staining with primary antibodies, nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Images were acquired using a Zeiss Axioskop 2 plus fluorescence microscope (Carl Zeiss, Jena, Germany).