Cy3 conjugated anti goat igg

The cells were grown onto poly (D-lysine)-coated cover glasses in a 24-well plate. After treatment, the cells were fixed for 20 min using cooled absolute ethyl alcohol, and then incubated with PBS containing 0.2% Triton X-100 for 30 min. After blocking with 5% non-fat milk in PBS for 1 h, the cells were then incubated with antibodies against GRP78 (1:500; Santa Cruz, sc-1050) or LC3 antibody (1: 400; MBL, PD 014) at 4°C for 24 h. The cells were sequentially incubated with Cy3-conjugated anti-goat IgG (1:400; Jackson ImmunoResearch, 705-166-147) and FITC-conjugated anti-rabbit IgG (1:400; Jackson ImmunoResearch, 111-095-003) for 2 h. Afterwards, the cells were incubated with 0.5 μg/ml 4,6-diamidino-2- phenylindole (DAPI, Sigma) for 10 min. Then the cells were mounted on slides and images of fluorescence were acquired using a laser confocal microscope (Carl Zeiss Microimage Inc., Thornwood, NY).

Mouse anti-Rac1 monoclonal antibody was purchased from Thermo Scientific. Goat anti-plectin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-phosphorylated FAK monoclonal antibody (anti-FAKp) was acquired from BD Transduction Laboratories™ (Franklin Lakes, New Jersey, USA). Secondary antibodies including Cy2-conjugated anti-mouse IgG and Cy3-conjugated anti-goat IgG used for immunofluorescence microscopy, horseradish peroxidase-conjugated goat and mouse antibodies used for Western blotting analysis, as well as biotin-conjugated goat and mouse antibodies used for immunohistochemistry were purchased from Jackson Immuno Research Laboratories (West Grove, PA, USA).

For assessment of somatotopic changes in motor neurons in the nucleus ambiguus, the brains were dissected and cryoprotected in 20% glycerin in PB and frozen on dry ice. Serial coronal frozen sections (40 µm thick) were obtained by using a sliding microtome (HM440E; Thermo scientific) and separated into 4 series^{56 (link)}. We used series 1 and 3 to avoid double-counting.
In the brain specimens from pyramidal decussation to facial nucleus, two series of sections were processed for immunofluorescence staining for anti-choline acetyltransferase (ChAT) to elucidate whether the FB and/or DY (FB/DY)-labelled neurons were motor neurons. In brief, the sections were blocked with 25% BlockAce in phosphate-buffered saline (PBS) (pH 7.4) containing 0.5% Triton-X and incubated in goat anti-ChAT antibody (1:200; Merck Millipore, Billerica, MA). Then, the sections were washed in PBS and incubated in Cy3-conjugated anti-goat IgG (1:400; Jackson ImmunoResearch, West Grove, PA). After washing, the sections were mounted, dried, and cover-slipped with DPX. Motor neurons of the nucleus ambiguus were identified based on ChAT immunoreactivity and the location in the reticular formation, and double-labelled (FB/DY and ChAT) neurons in the nucleus ambiguus were counted (Fig. 5d). As for the somatotopic changes, a distribution chart was created centering on the obex from every other section.

IF analysis was used to assess B7-H3 and CXCR4 colocalization in LV-B7-H3- or LV-NC-infected N87 cells. 1 × 10⁵ cells were seeded on glass coverslips in 24-well plates. After 1 d, cells were washed three times in PBS for 5 min/wash, fixed in 4% paraformaldehyde for 5 min, and washed again in PBS three times. Cells were treated with 0.25% Triton X-100 with 0.2% BSA for 5 min and then washed three times in PBS. Cells were blocked with 1% normal horse serum, and incubated with specific primary antibodies (B7-H3 or CXCR4) for 5 h at 4°C. After three PBS washes, cells were incubated with Cy3-conjugated anti-goat IgG (1:400; Jackson ImmunoResearch, USA) or Alexa Fluor 633-conjugated anti-rabbit IgG (1:400; Jackson ImmunoResearch, USA) for 1 h at 37°C, and then rinsed three times with PBS for 5 min/was. Nuclei were counterstained with DAPI (Sigma-Aldrich, USA) for 8 min, and slides were coverslipped. Cells were observed under a laser confocal microscope IX71 (Olympus, Japan) with a digital camera (Olympus, Japan).