Alexa fluor 568 goat anti mouse

Mice were sacrificed with cardiac perfusion by 4% paraformaldehyde (PFA). Before cardiac perfusion, mice were anaesthetized with 100-200 mg/kg body weight for Ketamine and 5-16 mg/kg body weight for Xylazine injected intraperitoneally. Brain was collected and post-fixed in 4% PFA for 24 h. Tissue was then embedded after dehydration with 30% of sucrose. Hippocampal and olfactory bulb cryosections were prepared with 20μm thickness. Briefly, sections after antigen retrieve (pH 6.0 citrate acid) and washing with 0.3% TritionX-100 PBS were incubated with 5% goat serum (0.3 TritionX-100). For BrdU staining, sections were incubated with 2M HCl for 1 h at room temperature (RT) and wash with PBST for 3 times (15min each time). Primary antibodies (Mouse-anti-BrdU, CST, 1:400; Rabbit-anti-DCX, CST, 1:400) were incubated with tissue sections at 4° C overnight. Secondary antibodies (Goat-anti-Rabbit-Alexa fluor 488, ThermoFisher, 1:800; Goat-anti-Mouse-Alexa fluor 568, ThermoFisher, 1:800) were incubated with sections for 2 h RT. DAPI (Sigma) was incubated with sections for 10min. Tissue IF images were obtained with confocal microscopy (Carl Zeiss, LSM 700) with Z-stack for 20μm. Image was shown as maximum projection of Z-stack. Required positive cell number was calculated and displayed as the cell density per mm².

The MCF-7 cells were plated onto coverslips at 2.5 × 10⁴/well and treated with tested drugs alone—TAM (20 µM) and ARA12 (25 μg/mL)—or in a combination with L. plantarum PM or L. rhamnosus PM in a concentration of 30% (v/v). After 48 h treatment, cells were fixed with 4% paraformaldehyde for 15 min. Then, cell membranes were permeabilized with 0.1% Triton X-100 for 10 min, following incubation with 2% goat serum in PBS for 1 h to block nonspecific binding sites. Afterward, the cells were incubated for 1 h with the primary antibody (1:250, mouse monoclonal anti-Ki-67 antibody, P6834, Sigma-Aldrich, St. Louis, MO, USA). For microscopy visualization, the cells were incubated with a secondary antibody conjugated with a fluorochrome (1:500, goat anti-mouse Alexa-Fluor568, Thermo Scientific, Rockford, IL, USA) for 1 h in the dark. Slides were mounted with ProLong Glass Antifade Mountant (Thermo Scientific, Rockford, IL, USA). Immunofluorescence results were examined using a fluorescence microscope and subjected to semiquantitative analysis based on the assessment of cells positive for Ki-67 expression (for each sample, signals from at least 100 cells were counted from 3 separate microphotographs performed with the same exposure time).

Rabbit and cynomolgus kidney sections were blocked with 5% goat serum (Vector Laboratories, UK) and incubated with mouse anti-fibronectin (Millipore; MAB88916-C) diluted 1:400 in PBS overnight at 4°C. Slides were washed five times in PBS and incubated with goat anti-mouse–Alexa Fluor 568 (Thermo Fisher Scientific; A11031) diluted 1:250, for 1 h at RT. Slides were washed five times in PBS and mounted in DAPI-Mowiol^®.

TA muscles were excised, embedded in OCT compound (23-730-571, Thermo Fisher Scientific), and frozen in liquid nitrogen–cooled 2-methylbutane. A cryostat (Leica CM 1950) was used to obtain 10 μm muscle sections onto microslides (48311-703, VWR). The slides were fixed in 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton X-100/1× PBS, and blocked in 0.5% BSA/10% goat serum/1× PBS for 20 minutes. The slides were incubated overnight with anti–xanthine oxidase antibody (1:100, rabbit monoclonal, ab109235, Abcam) and anti-CD31 antibody (1:100, mouse monoclonal, 66065-2-lg, Proteintech) in 0.5% BSA/2% goat serum/1× PBS. For the secondary antibodies, the slides were washed and incubated with goat anti-rabbit IgG (H+L) Alexa Fluor 647 and goat anti-mouse Alexa Fluor 568 (1:300, A-21244 and A-11004, Thermo Fisher Scientific) in the dark for 1 hour. The slides were washed and incubated with DAPI/1× PBS for 15 minutes to detect nuclei. Slides were mounted with SlowFade Diamond (S36967, Thermo Fisher Scientific) and imaged using a Keyence BZ-X910 at 20× original magnification. Image processing was done using ImageJ.

Anti-HA-Tag (C29F4) Rabbit mAb #3724 (dilution 1:3500), Anti-GAPDH (14C10) Rabbit mAb #2118 (1:5000), Anti-rabbit IgG, HRP-linked Antibody (1:5000) were obtained from Cell Signaling Technologies. Anti-DDX3 Rabbit Ab (1:5000, SAB3500206) was obtained from Sigma-Aldrich. Goat anti-rabbit-AlexaFluor⁴⁸⁸ (#a11034) and goat-anti mouse-AlexaFluor⁵⁶⁸ (#a11004) conjugates were purchased from Thermo-Fisher (dilution 1:1000). Anti-IFNAR antibody (#21385–1, PBL Interferon Source) and Isotype control IgG2a antibody (#554126, BD Pharmingen Product) were both used at 5 μg/ml. Anti-LASV (anti-GP: L-52-161-6, anti-NP: L-52-2159-15) and anti-JUNV (anti-GP: GD01, anti-NP: Y-MAO3-BE06) antibodies were obtained from the US Army research Institute of Infectious Diseases (USAMRIID) archives (PMID: 20686043, PMID: 22607481).

Slices were washed in phosphate-buffered saline (PBS) three times for 15 min and then blocked for 1 h in 0.5 mL of blocking solution (100 mM PBS, 0.005% bovine serum albumin, 0.4% Triton X-100, and 10% donkey serum). Slices were washed again and immersed in the primary antibody solution (10 mM PBS, 1% donkey serum, 0.1% sodium azide, 0.4% Triton X-100, and primary antibody) for 48 h at room temperature with continuous agitation. After a third washing step, slices were immersed in the secondary antibody solution (10 mM PBS, 1% serum, 0.1% sodium azide, 0.4% Triton X-100, and secondary antibody) and agitated continuously for 24 h. Slices were washed a final time with PBS, mounted onto 0.1% gelatin-subbed slides, embedded in DAPI-Fluoromount (Southern Biotech), and coverslipped. Details of the primary antibodies used are as follows: anti-GFP, chicken polyclonal (Aves, Davis, CA, USA, RRID:AB_10000240, 1:1000); anti-RGS14 monoclonal antibody (Neuromab, Davis, CA, USA, RRID:AB_10698026). Details of the secondary antibodies used are as follows (all diluted 1:1000): goat anti-chicken Alexa Fluor^® 555 (ThermoFisher Scientific, Waltham, MA, USA, A-21437, RRID:AB_2535858); goat anti-mouse Alexa Fluor^® 568 (Thermo Fisher Scientific, #A-21134, RRID:AB_2535773).

ERmoxGFP was a gift from Dr. Erik Snapp (Albert Einstein College of Medicine, presently at Howard Hughes Medical Institute, Janelia Research Campus, Virginia) (Addgene plasmid #68,072), and Sec61β-GFP from Dr. Gia Voeltz (University of Colorado, Boulder). Mouse anti-dsRNA antibody (Cat#: J2-1904) was purchased from Scions English and Scientific Consulting, goat anti-rabbit Alexa Fluor 532 from ThermoFisher Scientific (Cat#: A-11009), goat anti-mouse Alexa Fluor 568 from ThermoFisher Scientific (Cat#: A-11031), and goat serum from Thermo Fisher Scientific (Cat#: 16,210–064). Rabbit anti-NS2B (Cat#: GTX133308) and NS4B (Cat#: GTX133321) were kindly provided by Genetex (Cat#: GTX133321). 16% paraformaldehyde (Cat#: 15,710) and 25% glutaraldehyde (Cat#: 16,220) were from Electron Microscopy Sciences, USA. All other chemicals were obtained from Sigma. ZIKV strain (PRVABC59) was obtained from ATCC (Cat#: VR-1843). All virus manipulations were performed according to level 2 containment procedures (Dr. Jean; UBC B17-0024).