Alexa 594 conjugated goat anti mouse igg

CD147-specific antibody HAb18 was produced by our lab; α-tubulin antibody (sc-8035) was purchased from Santa Cruz; p190-A antibody (2513) was obtained from Cell Signaling Technology; p190-B antibody (611612) and paxillin antibody (610620) were obtained from BD biosciences; Alexa 594-conjugated goat anti-mouse IgG and Alexa Fluor 488 phalloidin were purchased from Invitrogen. A siRNA-resistant CD147 replacement vector was generated based on a previously reported CD147 expression plasmid [19 (link)] using the following primer: forward 5′-TCATGAACGGCTCCGAGAGCAGATTTTTTGTTTCATCCTCGCAGGGCCGGTCAGAGC-3′, reverse 5′-GCTCTGACCGGCCCTGCGAGGATGAAACAAAAAATCTGCTCTCGGAGCCGTTCATGA-3′. The RhoA biosensor was obtained from Addgene.

Small pulmonary arterial wall thickness was measured by immunofluorescence staining of lung sections with antibody to α-smooth muscle actin (αSMA). Formalin-fixed and paraffin-embedded sections were deparaffinized in graded ethanol solutions. Antigenic sites of the lung tissue were unmasked by boiling in Na-citrate buffer (pH 5.5) using a pressure cooker. Immunofluorescence staining was performed with mouse anti-human αSMA primary antibody (clone 1A4, 1∶400; Sigma, St. Louis, MO) followed by Alexa594-conjugated goat anti-mouse IgG (1∶400; Invitrogen, Carlsbad, CA). Stained lung sections were scanned with Aperio ScanScope FL at 20× magnification. Wall thickness was assessed in αSMA positive staining in vessels (20–100 µm in diameter; n = 80–100 vessels) for each animal (n = 3 for each group). The wall thickness was measured using Aperio ImageScope software and calculated using the following equation:
Wall thickness  =  Wall width/Vessel width.

The neurons and glial cells were immunocytochemically distinguished after fluorescence imaging. Antibodies for gamma amino butyric acid (GABA) and glial fibrillary acidic protein (GFAP) were used for identification of the neurons and glial cells, respectively. Cells were fixed with 4% paraformaldehyde (PFA) for 30 min. Fixed cells were immunized with rabbit anti-GABA polyclonal antibody (1:1000 dilution; Millipore) and mouse anti-GFAP monoclonal antibody (1:1000 dilution; Sigma-Aldrich) in PBS including 0.1% Triton X-100 and 1% goat serum for 2 days in 4 °C. Alexa 488-conjugated goat anti-rabbit IgG and Alexa 594-conjugated goat anti-mouse IgG (1:200 dilution; Invitrogen) were used as the second antibody for GABA and GFAP, respectively. GABA- and GFAP-positive fluorescent signals were visualized with inverted fluorescent microscopy (BioRevo, Keyence).

Immunostaining was performed as described (Nikitina et al., 2009 ) with slight modifications; embryos were incubated overnight at 4 °C in primary antibody in blocking solution (10% sheep serum in PBS with 0.1% Triton X-100); secondary antibodies were also incubated overnight at 4 °C. Histochemical reactions were performed as described (Patel, 1994 (link)). Before imaging, embryos were cleared through a glycerol series into 70% glycerol in PBS. Primary antibodies: 1:50 HNK1 (mouse IgM, clone 3H5, Developmental Studies Hybridoma Bank); 1:500 anti-HuC/D (mouse IgG2b; Invitrogen); 1:200 anti-neurofilament-M (mouse IgG2a; Invitrogen); 1:200 anti-Pax3/7 (clone DP312; Davis et al., 2005 (link)). (The Developmental Studies Hybridoma Bank was developed under the auspices of the NICHD and is maintained by the University of Iowa, Department of Biological Sciences, Iowa City.) Secondary antibodies: 1:1000 Alexa⁴⁸⁸-conjugated goat anti-mouse IgG and/or Alexa⁵⁹⁴-conjugated goat anti-mouse IgG (Invitrogen), or 1:600 horseradish peroxidase-conjugated or alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch).

Cells on coverslips were harvested 8hs post infection (HPI) or 48hs after
transfection, fixed in 4% paraformaldehyde for 5 min, and then permeabilized
with phosphate buffered saline (PBS) with 0.1% Triton X-100 for 20 min at room
temperature. The coverslips were then incubated overnight at 4°C with primary
antibodies (S1
Table). After rinsing, cells were incubated for 30 min with Alexa
594-conjugated goat anti-mouse IgG and Alexa 488-conjugated goat anti-rabbit IgG
(Invitrogen, Carlsbad, CA), and then counterstained with
4',6-diamidino-2-phenylindole (DAPI). Images were captured using a confocal
laser microscope system (Leica TCS SP5, Leica Microsystems, Wetzlar, Germany). A
sequential multiple fluorescence scanning mode was used to avoid nonspecific
overlap of signals. In some experiments, manual counting of infected cells was
carried out in five different regions of the coverslips.