Cy3 labeled goat anti mouse igg

Immunofluorescence (IF) assay was used to assess the expression of WT1, LC3II and nephrin protein in kidney tissues. A total of 0.25% Triton X-100 was used to permeabilize 5-μm thick kidney sections for 15 min, and 5% Bovine Serum Albumin (BSA) was used to block them for 1 h at room temperature. Then, the sections were stained with primary antibodies against WT1 (1:200), LC3II (1:200), and nephrin (1:500) overnight at 4°C, followed by secondary Cy3-labeled goat anti-rabbit IgG (A0516, Beyotime), Cy3-labeled goat anti-mouse IgG (A0521, Beyotime) or FITC-labeled goat anti-rabbit IgG (A0561, Beyotime). The cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI), and images were obtained with fluorescence microscopy.

HeLa cells were grown on coverslips overnight and infected with NDV strain Herts/33 at an MOI of 1 or stimulated with poly (I:C) for 12 h. Cells were fixed in 3% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 10 min, incubated in blocking buffer (3% BSA/PBS), and then stained with mouse mAb J2 (Scicons, Hungary) followed by Cy3-labeled goat anti-mouse IgG (Beyotime). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific Inc., Rockford, IL, USA) at a dilution of 1:500 for 10 min, and then the coverslips were mounted on slide glasses and visualized using a fluorescence microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan).

The previously stored MAC-T cells (17 (link)) in liquid nitrogen were resuscitated and cultured with 10 mL Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) medium (Hyclone, UT, USA) containing 10% fetal bovine serum (System Biosciences, Mountain View, CA, USA) in a 5% CO₂ humidified incubator at 37°C and then passaged every 2 days.
MAC-T cells were identified by immunofluorescence analysis with epithelial marker cytokeratin 18 (18 (link)). The cells were inoculated in a six-well plate (NEST, Wuxi, Jiangsu, China). When the cell confluence was 80%, the medium was discarded. The cells were rinsed three times with PBS, and fixed at room temperature for 20 min with pre-cooled 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), then added 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), and incubated at room temperature for 5 min. After blocking for 1 h at room temperature in 2% BSA. the cells were incubated with primary antibody for cytokeratin 18 (1:500, Santa Cruz, Dallas, TX, USA) overnight at 4°C. The cells was incubated with Cy3-labeled goat anti-mouse IgG (1:200, Beyotime, Shanghai, China) for 1 h at room temperature, then incubated with DAPI (Beyotime, Shanghai, China) for 10 min at room temperature, followed by seal the coverslip and imaging under fluorescence inverted microscope (Olympus, Shinjuku-ku, Tokyo, Japan).

Immunofluorescence in regards to the frozen placental tissues of the third trimester, detection of PFKFB3 activity in the normotensive and preeclamptic placenta was performed as described previously [25 (link)]. Briefly, the primary antibodies were diluted in the appropriate blocking solution at the following concentrations: PFKFB3 antibody (1: 100; 13763-1-AP, Proteintech) and mouse anti-CK7 (1: 100; ab68459, Abcam). The secondary antibody was a FITC-labeled goat anti-rabbit IgG (Beyotime) and a Cy3-labeled goat anti-mouse IgG (Beyotime). The nuclei were stained with DAPI (Beyotime) for 3 min. HTR-8/SVneo cells in different groups cultured on coverslips were fixed by 4% paraformaldehyde for 20 min and then extracted with 0.5% Triton X-100 solution for 5 minutes. After blocking with TBST containing 1% bovine serum albumin, cells were incubated with indicated primary antibody NF-κB1 (1: 100; 14220-1-AP, Proteintech) for 1 h. After that, cells were washed and incubated with FITC-labeled goat anti-rabbit IgG (Beyotime) for 1 h, following with DAPI for 3 min. Images were captured with an inverted microscope and were analyzed by ImageJ.

Brains were dehydrated in graded alcohol and embedded in paraffin. The brains were cut into 5 μm thick coronal sections. Slices were blocked with 5% goat serum for 2 h and incubated with rabbit polyclonal anti-COX2 antibody (#ab15191, 1:200, Abcam) and mouse monoclonal anti-NeuN antibody (#GTX30773, 1:200, GeneTex, USA) overnight at 4 °C. The sections were incubated with Alexa 488-labeled goat anti-rabbit IgG (#A0423, 1:200, Beyotime, China) and Cy3-labeled goat anti-mouse IgG (#A0521, 1:200, Beyotime) for 1 h at 37 °C. The nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI, Beyotime) for 5 min at room temperature. The cells were imaged by an Olympus fluorescence microscope.
Neurons were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 in PBS and blocked with 5% goat serum for 2 h. The remaining steps were consistent with the method mentioned above.

Mv.1.Lu cells were cultivated on cover glasses in 24-well plates, followed by infection with PS or CDV₃ at an MOI of 2 when the cells reached ~70% confluence. The mock-infected cells were treated with PBS as a negative control. Next, at 24 hpi, the cells were fixed with 4% paraformaldehyde and subsequently permeabilized with 0.1% Triton X-100. Further, the cells were incubated with an NF-κB P65 rabbit polyclonal antibody (Beyotime, China) and a mouse monoclonal antibody specific to CDV N protein and incubated with Cy3-labeled goat anti mouse IgG (Beyotime, China) and FITC-conjugated goat anti-rabbit IgG secondary antibody (ThermoFisher, U.S.A) prior to staining with DAPI. The fluorescent images were analyzed under confocal microscopy (Leica, Germany).