Alexa fluor 647 donkey anti goat igg

hiPSCs or kidney organoids were fixed with 4% paraformaldehyde. The following antibodies were used: NANOG (1E6C4, 1:100; Santa Cruz Biotechnology), SSEA-4 (813-70, 1:100; Santa Cruz Biotechnology), TRA-1-81 (TRA-1-80, 1:100; Santa Cruz Biotechnology), biotinylated Lotus Lectin (LTL, B-1323,1:100; Vector Laboratories), E-Cadherin (ab11512, 1:50; Abcam), CD77 (551352, 1:100; BD Biosciences), and Podocalyxin (BAF1658, 1:100; R&D Systems). Antibody staining was visualized using the following secondary antibodies: Alexa Fluor 488-donkey anti-rat IgG (1:250; Invitrogen), Alexa Fluor 488-donkey anti-mouse IgG (1:250; Invitrogen), Alexa Fluor 647-donkey anti-goat IgG (1:250; Invitrogen), or Cy3-streptavidin (1:1000; Jackson ImmunoResearch). Cell nuclei were then counterstained with 4,6-diamidino-2-phenylindole (DAPI; Roche, Mannheim, Germany). Stained sections were observed using a confocal microscope (LSM700; Carl Zeiss Co. Ltd., Oberkochen, Germany). Images were converted to TIFF format and contrast levels were adjusted using Adobe Photoshop v. 13 (Adobe System, San Jose, CA, USA).

For immunohistochemical studies, formalin-fixed, paraffin-embedded (FFPE) mouse liver samples were sectioned at 4 μm, deparaffinized, and subsequently stained with anti-GFP (1:200, CST, Cat. #2956) or anti-Fah antibody (1:100, Abcam, Cat. #83770). Visualization was performed using the DAB Quanto kit (Fisher Scientific, Cat. # TA-125-QHDX) as instructed by the manufacturer.
For immunofluorescence, N2A cells grown on coverslides were fixed with 4% paraformaldehyde for 15 min at room temperature (RT) and permeabilized with 0.1% Triton X-100/PBS at RT for 15 min. Cells were then incubated overnight at 4 °C with anti-streptavidin antibody (1:100, Vector, Cat. # BA-0500-.5) and 1 h at room temperature with Alexa Fluor 647 Donkey anti-goat IgG (Invitrogen, Cat. #A32849). Nuclei were counterstained with DAPI. Images were acquired on a Leica DMi8 imaging microscope.

Wild-type HeLa cells were seeded on coverslips and treated with 300 nM thapsigargin (Tg) for 30 min, 4 or 16 h, respectively. Cells were fixed with 2% (w/v) paraformaldehyde at room temperature for 20 min, permeabilized by incubation in 0.2% (v/v) Triton X-100/PBS for 5 min and incubated in blocking solution (5% (w/v) BSA, 0.1% (v/v) Tween-20 in PBS, sterile-filtered) for 30 min at room temperature. Primary antibodies were dissolved in blocking solution and added to the coverslips for 1 h. The following antibodies and dilutions were used: RTCB (Santa Cruz, 1:500), archease (monoclonal, 1:2) and calnexin (Santa Cruz, 1:100). The archease monoclonal antibody was generated by immunizing mice with wild-type histidine-tagged archease purified as described previously (Popow et al, 2014 (link)). Cells were washed three times in 0.1% PBST and incubated with fluorescent secondary antibodies diluted in blocking solution for 1 h: Alexa Fluor® 488 donkey anti-rabbit IgG (Invitrogen, 1:500), Alexa Fluor® 568 donkey anti-mouse IgG (Invitrogen, 1:500), and Alexa Fluor® 647 donkey anti-goat IgG (Invitrogen, 1:500). Coverslips were again washed four times with 0.1% PBST and subsequently mounted in ProLong® Gold Antifade Mountant with DAPI (Invitrogen). Images were taken at least 24 h after mounting using a laser-scanning confocal microscope (LSM780, Zeiss).

For immunofluorescence, HeLa cells were cultured on coverslips in 24 well plates overnight and then transfected with N, P, VIM and VIM mutant plasmids. HPIV3 and HPIV3_HA-P were added before transfected. Cells were harvested at the indicated times. 4% paraformaldehyde in 1×PBS was used to fix cells and 0.2% Triton X-100 were added for 20min at room temperature. After being blocked with 3% bovine serum albumin (BSA), primary antibodies diluted in 1% BSA were added and then secondary antibodies diluted in 1% BSA were added at 4°C.The primary antibodies used here were as follows: goat anti-HPIV3 (Abcam), rabbit anti-Flag tag (CST), rat APC-anti-Flag (Biolegend), mouse anti-c-Myc tag (MBL), mouse anti-HA tag (sigma), rabbit anti-HA tag (CST).The secondary antibodies used here were as follows: Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen), Alexa Fluor 594 donkey anti-mouse IgG (Invitrogen), Alexa Fluor 647 donkey anti-goat IgG (Invitrogen).Image J software were used to assay the fluorescence intensity of the cells in drawing boxes and intensity values were used. In order to visualizing the IBs in cells, HPIV3_HA-P virus was used and the location of HA-P was considered as viral IBs. HA-P and Myc-N co-expression also formed viral IBs in cells without HPIV3 infection. To counting the numbers of large, medium and small viral IBs, almost 30 cells in each group with IBs were counted.