Cy3 conjugated secondary antibody

The sciatic nerves were isolated and fixed in 4% PFA for 24 h, then the tissues were cryoprotected in a 20% sucrose solution. Cross-serial sections (10 μm) were prepared using a cryostat (Leica, Nussloch, Germany); then, the slides were permeabilized with cold methanol and washed with PBS followed by blocking with 5% FBS and 0.3% Triton X-100 in PBS for 1 h. The sections were incubated with a primary antibody against MPZ (Abcam, Cambridge, UK) or NCAM (R & D Systems, Minneapolis, MN, USA) for 16 h at 4 °C, followed by incubation with Alexa 488 or Cy3-conjugated secondary antibody (Sigma) for 2 h. After coverslips adhered to the glass slides with mounting medium (Biomeda, Foster City, CA, USA), images were obtained under a Zeiss imager M2 with an ApoTome II microscope (Carl Zeiss). The intensity of the immunofluorescent staining was measured over an area of 500 × 600 μm² using Zen 2 Blue edition software.

For 2D SMCs, cells were cultured on Lab-Tek chamber slides (Thermo Scientific). The indirect immunostainings for both spheroids and 2D SMCs were performed on day 2 of culture. Samples were stained at 4°C overnight using the following primary antibodies for the contractile SMC proteins: anti-α-smooth muscle actin (α-SMA) (Sigma, A5228, 1 : 200), anti-calponin (Sigma, C2687, 1 : 100), anti-smoothelin (Novus, NBP2-37931, 1 : 100), and anti-myosin heavy chain 11 (MyH11) (Santa Cruz, SC-6956, 1 : 50). The slides were incubated with a Cy3-conjugated secondary antibody (Sigma, 1 : 500) at room temperature for 1 h.
For ECM proteins, anti-collagen I (Abcam, ab260043, 1 : 150), anti-collagen III (Abcam, ab7778, 1 : 150), anti-fibronectin (Santa Cruz, sc-59826, 1 : 300), and anti-elastin (Abcam, ab9519, 1 : 100) were used. Samples were stained at 37°C for 4 h. The slides were incubated with a FITC-conjugated secondary antibody (Sigma, 1 : 500) at 4°C overnight. The slides were counterstained with DAPI (4′,6-diamidino-2-phenylindole, Sigma, 1 : 400) and analyzed with an inverted confocal microscope (Leica DMI6000 AFC, Model SP8). For negative controls, the primary antibody was omitted. The specificity of the commercial antibodies had been thoroughly validated in our previous studies [28 (link), 29 (link)].

The 5-μm-thick paraffin sections were cut and fluorescent immunohistochemical analysis of sections was performed as previously described [24 (link)]. Cell infiltration in the skeletal muscles was examined by using anti-mouse Mac-3 antibody (1:200; Cedarlane Labs, Burlington, ON, Canada) overnight at 4 °C followed by incubation with a Cy3-conjugated secondary antibody (1:500; Chemicon International, Temecula, CA, USA). The slides were then washed in PBS and stained with anti-sarcomeric α-actin antibody (1:200; Abcam Inc. Cambridge, MA, USA) and incubated with an FITC-conjugated secondary antibody (1:500; Chemicon International). The nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA, USA). Capillary density from wild type and ECKO mice was assessed by immunostaining with anti-platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) antibody (1:200, LifeSpan BioSciences, Nottingham, UK) overnight at 4 °C and then with a FITC-conjugated secondary antibody (Chemicon International). Digital images were captured with a Nikon digital camera mounted directly onto a fluorescence microscopy. Images were analyzed with computerized imaging software (NIS-Elements AR 3.10, Nikon Instruments Inc., Melville, NY, USA) as previously described [24 (link)].

Immunohistochemical analysis of lung tissues was performed with primary antibodies against α-smooth muscle (SM)-actin (Sigma-Aldrich) and Notch3 ICD (Abcam) using the Dako LSAB peroxidase kit (Dako). Staining of α-SM-actin was used to indicate the medial layer of small pulmonary arteries (PAs) for the assessment of medical wall thickness (MWT). For the Notch3 signal, lung tissue sections were incubated with rabbit anti-Notch3 and mouse anti-α-SM-actin antibodies for 1 hour, followed by incubation with Alexa-488-conjugated secondary antibody (green, Invitrogen) for Notch3 or Cy3-conjugated secondary antibody (red, Chemicon) for α-SM-actin at room temperature for 30 minutes, and observed with a Leica TCS SP spectral confocal microscope at Microscope Core Laboratory of Chang Gung Memorial Hospital.

The brain was perfused with cold saline and fixed with phosphate buffer (pH 7.4) containing 4% formaldehyde. Coronal brain sections (8 μm thick) were incubated with blocking buffer (4% w/v, Block Ace; Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan) for 2 h. The sections were incubated with polyclonal rabbit anti-cleaved caspase-3 or RIP3 antibody (1 : 100, Abcam, Cambridge, UK) in 1% w/v Block Ace overnight at 4°C. After washing with PBS, these were correspondingly incubated with Cy3-conjugated secondary antibody (1 : 100, Chemicon, Temecula, CA, USA) for 2 h at room temperature. Finally, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA) for 15 min at room temperature, washed, and mounted using a mounting medium with 80% glycerol. Immunofluorescence was visualized using the fluorescence microscopy as described above. The percentage of cleaved caspase-3-positive cells was calculated as the number of the positive-staining nuclei to the total nuclei [33 (link), 40 (link)]. Regarding RIP3, because a lower level of expression of this protein was observed in every cell in the hippocampus, the cells having an enhanced expression of RIP3 under a laser beam at constant intensity were determined as RIP3-positive. All histopathological scoring and evaluation was performed by an investigator without knowledge of the treatment.

Untransfected, EGFP, EGFP-Tir, EGFP-EspF and EGFP-Map cells were cultured on glass cover-slips until confluent and fixed with chilled methanol for 5 minutes at −20 °C, rehydrated with PBS for 5 minutes at room temperature and blocked with PBS containing 0.5% BSA at room temperature for 30 minutes. Cover slips were incubated with primary antibodies against TJ proteins for 2 to 4 hours. Primary antibodies, used at 1:300 dilution, were: claudin-1 (Thermo Fisher Scientific, #374900, mouse monoclonal), claudin-4 (Thermo Fisher Scientific, #329400, mouse monoclonal), occludin (Thermo Fisher Scientific, #331500, mouse monoclonal) and ZO-1 (Thermo Fisher Scientific, #339100, mouse monoclonal). After washing three times with blocking solution, the cover slips were incubated with Cy3-conjugated secondary antibody (Millipore) for 1 hour at room temperature. Cover-slips were mounted in ProLong Diamond Antifade mountant (Thermo Fisher Scientific, #P36961). Images were acquired at 63X magnification on a Zeiss ApoTome (Axiovert 40 CFL).