Cy3 conjugated goat anti rabbit igg

PDL cells were fixed with 4% paraformaldehyde in PBS pH 7.4 for 10 min, washed with PBS (Sigma-Aldrich, Munich, Germany), and treated with 0.1% Triton X-100 (Sigma-Aldrich) for 5 min, as previously reported [45 , 46 ]. Then, cells were washed again and blocked with nonfat dry milk (Bio-Rad Laboratories) for 1 h. After washing, cells were incubated with a primary rabbit anti-SMAD1/5/8 antibody (1 : 200; Santa Cruz Biotechnology, Santa Cruz, CA, Germany) for 90 min and with CY3-conjugated goat anti-rabbit IgG (1 : 2,000; Abcam, Cambridge, MA, USA) for 45 min. The SMAD1/5/8 nuclear translocation was analyzed using an Axioplan 2 imaging microscope (20x objective; Carl Zeiss). Images were captured with a PVCAM camera and the VisiView capturing software (Visitron Systems, Puchheim, Germany).

Pathology was performed on mice sacrificed on day 5 post infection. The lung samples were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 3.5 μm sections. The fixed tissue samples were stained with hematoxylin–eosin (H&E), and the SARS-CoV-2 antigen was detected by indirect immunofluorescence (IFA). The tissue sections were stained with H&E for routine histology. For IFA, the slides were dewaxed and rehydrated, followed by thermally induced antigen repair within 15 min in the microwave with ethylenediamine tetraacetic acid. The slides were washed with PBS and 0.02% Triton X-100 and then sealed with 5% BSA for 1 h at room temperature. Primary antibody (rabbit anti-SARS-CoV-2 N protein polyclonal antibody, made in-house: 1:500) was dropped onto the slides and then washed in PBS. When the slide was nearly dry, the tissue was covered with Cy3-conjugated goat anti-rabbit IgG (ABCAM, AB6939) at a 1:200 dilution. After washing with PBS, the slides were stained with DAPI (Beyotime) at a 1:100 dilution. The image information was collected using a Pannoramic MIDI system (3DHISTECH, Budapest).

Animal necropsies were performed according to a standard protocol. Tissues for histological examination were stored in 10% neutral-buffered formalin for 7 days, paraffin-embedded, sectioned, and stained with hematoxylin and eosin prior to examination by light microscopy. To examine the SARS-CoV-2 antigen, paraffin dehydrated tissue sections were placed in antigen repair buffer for antigen retrieval in a microwave oven. The tissue was blocked with 5% BSA at room temperature for 1 h, followed by incubation with house-made primary antibody at 1:500 (rabbit anti-CoV N protein polyclonal antibody). After being washed by PBS, the slices were slightly dried and covered with Cy3-conjugated goat anti-rabbit IgG (Abcam) at 1:200 dilution. The slides were stained with DAPI (5 µg/mL) after washing by PBS. The image was collected by Pannoramic MIDI system (3DHISTECH, Budapest, Hungary).

Cells were fixed with 4% paraformaldehyde for 15 min. Next, Triton X-100 (0.1%) was used to incubate cells for 30 min. Cell block was conducted with 1% BSA for 15 min. All these operations were performed at room temperature. Primary antibody incubation was performed with Nrf2 antibody (1:100 diluted in PBS) (AF0639, Affinity) overnight at 4 °C. After washing three times with PBS, cells were incubated with Cy3-conjugated goat anti-rabbit IgG (1:200 diluted in PBS) (ab6939, Abcam, UK) for 1 h at room temperature in a dark environment. Finally, nuclei were stained with DAPI. Staining was observed under a BX53 microscope (Olympus).

Midguts from infected mosquitoes were dissected (n = 30) at 14 dpi. The tissues were fixed using 4% paraformaldehyde for 1 h and washed with PBS containing 0.3% Triton X-100 (PBST) five times. Next, tissues were placed in blocking solution (PBS containing 5% goat serum and 0.3% Triton X-100) for 1 h and then incubated with primary rabbit anti-TIBOV-VP7 antibody (derived from rabbit serum, diluted 1:200 in PBST containing 5% goat serum) for 24 h, followed by secondary Cy3-conjugated goat anti-rabbit IgG (diluted 1:250 in PBST containing 5% goat serum; Abcam) for 12 h. The actin cytoskeleton was stained with Alexa Fluor 488 Phalloidin (Invitrogen) for 1 h. After each step, tissues were washed at least five times in 0.3% PBST to prevent the effects of reagents from affecting subsequent operations. Finally, tissues were mounted onto slides using SlowFade Diamond Antifade Mountant (Invitrogen), and images were recorded through a Leica SP8 confocal microscope (filter information TD 458/514/561, Leica, Germany). Using LAS X software (Leica), z-stack images were merged, and scale bars were added. PowerPoint 2019 was utilized for image grouping. All samples were analyzed under the same microscope and software settings.

HeLa cells transiently expressing ACE2 were prepared using Lipofectamine 3000 (Thermo Fisher Scientific) in a 96-well plate; mock-transfected cells were used as controls. 2019-nCoV grown in Vero E6 cells was used for infection at a MOI of 0.5. APN and DPP4 were analysed in the same way. The inoculum was removed after absorption for 1 h and washed twice with PBS and supplemented with medium. At 24 h after infection, cells were washed with PBS and fixed with 4% formaldehyde in PBS (pH 7.4) for 20 min at room temperature. ACE2 expression was detected using a mouse anti-S tag monoclonal antibody and a FITC-labelled goat anti-mouse IgG H&L (Abcam, ab96879). Viral replication was detected using a rabbit antibody against the Rp3 N protein (generated in-house, 1:1,000) and a Cy3-conjugated goat anti-rabbit IgG (1:200, Abcam, ab6939). Nuclei were stained with DAPI (Beyotime). Staining patterns were examined using confocal microscopy on a FV1200 microscope (Olympus).