Texas red goat anti rabbit

A72 cells were infected or not with CCoV at a MOI of five, and exposed or or not to OMF (0.5 µM). At 24 h p.i., IF staining was assessed [17 (link),52 (link)] using antibodies dissolved in 5% bovine serum albumin-TBST: anti-AhR (Sigma-Aldrich) (1:250); anti-NP monoclonal mouse, MAB 938 (The Native Antigen Company, Kidlington, UK); Alexa Fluor 488 goat anti-mouse (Thermo Fisher Scientific, Waltham, MA, USA) (1:1000); and Texas Red goat anti-rabbit (Thermo Fisher Scientific) (1:100). Microscopic study and photography were undertaken using a ZOE Fluorescent Cell Imager (Bio-Rad Laboratories). Fluorescence signals of images from microscopy were quantified using ImageJ software (version 1.53a, National Institutes of Health, Bethesda, MD, USA).

Monolayers of A72, pretreated or not with CH223191, were infected or not with CCoV, at an MOI of 0.05 and 5, and incubated at 37 °C. After 24 h p.i., immunofluorescence staining was evaluated as previously reported [21 (link),37 (link)], by using the following antibodies, diluted in 5% bovine serum albumin-1X Tris-Buffered Saline, 0.1% Tween^® 20 Detergent: anti-AhR (Sigma-Aldrich, St. Louis, MI, USA) (1:250), anti-NP monoclonal mouse, MAB 938 (The Native Antigen Company, Kidlington, UK), Alexa Fluor 488 goat anti-mouse (Thermo Fisher Scientific, Waltham, MA, USA) (1:1000), and Texas Red goat anti-rabbit (Thermo Fisher Scientific, Waltham, MA, USA) (1:100). Microscopy and photography were assessed by ZOE Fluorescent Cell Imager (Bio-Rad Laboratories, Hercules, CA, USA). Quantification of fluorescence signals from microscopy-generated images was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA) software.

S2 cells (1.5 × 10⁶) were transfected for 48 h with 2 μg of pAc5.1/V5-HisB plasmid constructs expressing N- or C-terminally HA-tagged ORFx. Cells were seeded onto coverslips and fixed with 3% paraformaldehyde for 15 min. Subsequently, cells were permeabilized with 0.2% Triton X-100 in 1× PBS for 1 h before blocking coverslips with 3% BSA in 1× PBS for 1 h. Coverslips were then incubated with rabbit anti-HA (1:1000; Cell Signaling) and either mouse anti-Lamin (1:1000; DSHB), mouse anti-Golgin84 (1:1000, DSHB), or mouse anti-Calnexin 99A (1:1000, DSHB) in blocking solution overnight at 4°C. Cells were washed three times with 1× PBS and slides were incubated with secondary antibodies goat anti-mouse Alexa Fluor 488 (1:5000; Thermo Fisher) or goat anti-rabbit Texas Red (1:5000; Thermo Fisher) for 1 h at room temperature. Cells were then washed two times with 1× PBS before being stained with Hoechst dye in PBS (1:10 000; Sigma Aldrich) for 10 min and washed once more. Finally, slides were analyzed using a Leica SP5 confocal microscope with a 63× oil objective lens and a 2× digital zoom. Z-stacks of 15 slices each were taken of for each condition.

MDA-MB-231 cells were treated with 10 mM HPβCD for 24 h at 37 °C. Following treatment, the media was removed, and cells were washed with PBS (Thermo Fisher Scientific, USA). Cells were subsequently fixed using 4% paraformaldehyde (Sigma Aldrich, UK) at room temperature for 10 min. Cells were then permeabilized by incubating in 0.1% Triton-X (Sigma Aldrich, UK) for 15 min. Thereafter a one-hour blocking step was completed by incubating cells with 1% bovine serum albumin (BSA) (VWR Life Sciences, Radnor, PA, USA) made up in 0.1% PBS-Tween 20 (PBST). Following this, primary antibody was added (Rabbit anti-human SFRP1 (Abcam Boston, MA, USA); (ab4193—1:250 dilution)) and incubated at 4 °C overnight. Once the incubation was complete, the secondary antibody was subsequently added (Goat Anti-Rabbit Texas Red (Thermo Fisher Scientific, USA); (T-2767—1:1000 dilution)) and incubated for a duration of 45 min. For nuclear staining, 0.0001 mg/mL DAPI (Sigma Aldrich, UK) was administered. Following the addition of ProLong™ Gold Antifade mounting media (Thermo Fisher Scientific, USA) to the slides, cells were subsequently visualized by using the Floid™ Cell Imaging System (Thermo Fisher Scientific, USA). Immunofluorescence intensity was quantified by utilizing the ImageJ software (Software Version 1.41) (NIH, Bethesda, MD, USA).

Chips were manually rinsed with pre-warmed DPBS, fixed with 4 % paraformaldehyde (PFA) (Merck) for 30 min and washed again with DPBS before permeabilisation in immunofluorescence wash buffer (IF buffer [17 (link)]) containing 0.1 % Triton X-100 for 15 min. Chips were blocked for 1 h at room temperature in DPBS/5 % normal goat serum/0.1 % Triton X-100 (Merck). Primary antibodies, diluted 1/100 in blocking buffer, were applied to cells and incubated overnight at 4°C. Chips were washed three times with IF buffer and secondary antibody (diluted 1/200 in blocking buffer) applied for 1 h at room temperature in the dark. Cells were again rinsed three times with IF buffer before staining nuclei with NucBlue DNA stain (2 drops per mL) (Thermo Fisher Scientific) in IF buffer. This was removed and replaced with DPBS for imaging on a Zeiss LSM 710 confocal microscope, processing images using ImageJ [18 (link)]. Antibodies were as follows: EpCAM (orb10618, Biorbyt, Cambridge, UK), cluster of differentiation 31 (CD31) Clone HEC7 (MA3100, Thermo Fisher Scientific), ZO-1 (INV 40-2200, Thermo Fisher Scientific), β-catenin (8480T, Cell Signalling Technologies, Danvers, MA). Secondary antibodies were goat anti-mouse FITC-conjugated and goat anti-rabbit Texas red, both from Thermo Fisher Scientific.