Alexa 546 labeled secondary antibody

Platelets and CD34 + -derived differentiated MK were allowed to adhere over coated fibrinogen (100 µg/ml) for 1 hour and 3 days respectively. Platelets, MK and bone marrow smears were fixed in 1% paraformaldehyde for 10 min at room temperature. After washing, cells were permeabilized with 0.3% Triton X100 in PBS for 5 min, blocked using 3% BSA PBS for 1 hour and incubated overnight with rabbit anti-CSTA antibody (Genetex; 63944). Next, cells were incubated with anti-rabbit Alexa-546-labeled secondary antibody (Life technologies; A11010), Alexa-488-labeled Phalloidin (Life technologies; A12379). Finally, after washing steps, the slides were mounted with DAPI-containing Fluoromount and examined using an AXIO Imager M1 microscope (Carl Zeiss, Germany).

All tissue samples were immersion-fixed in 10% neutral buffered formalin for at least 7 days under BSL4 conditions. Prior to removal from the BSL4 laboratory, formalin was changed and specimens were processed under BSL2 conditions by conventional methods, either embedded in paraffin, sectioned at 5 µm thickness and stained with hematoxylin and eosin (H&E) or embedded in Tissue Tek and frozen sections cut at 3–8 µm thickness and used for immunofluorescent (IF) staining. Tissues for immunohistochemistry (IHC) were stained as previously described using a rabbit anti-NiV-nucleoprotein (N) antibody (kindly provided by Dr. C. Broder, Uniformed Services University, Bethesda, MD) [13] (link). Tissues for IF were stained with a rabbit anti-NiV-N antibody, a biotinylated anti-CD31 (eBioscience), anti-collagen IV labeled with Alexa 647 (eBioscience), anti ephrin B2 (Santa Cruz Biotechnology) or ephrin B3 (R&D Systems) and Hoechst for nuclear staining. NiV N in mouse tissue could only be detected following immunofluorescent staining, likely due to the limit of detection by conventional IHC. An Alexa 546 labeled secondary antibody (Life Technologies) was used for detection of the anti NiV N antibody as well as anti ephrin B2 and B3 antibodies and an Alexa 488 conjugated streptavidin (R&D Systems) was used for detection of the anti-CD31 antibody.

The immunochemical detection of PAR formation was performed as previously described [28 (link),31 (link)]. HaCaT cells were seeded at 0.3 × 10⁶ cells per well in 12-well plates, on sterile 18 mm glass coverslips 24 h before the treatment. Directly after the toxic exposure, cells were fixed with ice-cold methanol for 7 min at −20 °C. Next, samples were washed 3 × 10 min with PBS and permeabilized with 0.4% Triton X-100 in PBS for 3 min. Then, samples were incubated in blocking solution (BS), containing 5% (w/v) milk powder and 0.5% Tween-20 in PBS, for 1 h at RT, and incubated with anti-PAR antibody (10H) (1:300 in BS) [36 (link)] overnight at 4 °C in a humidified chamber. Next, after 3 × 10 min washing with PBS, coverslips were incubated with Alexa546-labeled secondary antibody (Invitrogen) (1:300 in BS) for 1 h at RT in the dark in a humidified chamber. To visualize nuclei, samples were washed 3 × 10 min with PBS and incubated with Hoechst 33342 dye (0.2 µg/mL in PBS) (Thermo Fischer Scientific) for 5 min at RT. Next, coverslips were washed 3 × 10 min with PBS and mounted on glass slides using Aqua PolyMount (Polysciences, Warrington, PA, USA). Images were acquired using the Zeiss Axio Obrerver Z1 epifluorescence microscope and Axiovision software. At least 200 nuclei per sample were automatically analyzed using KNIME v.4.5.0 software [37 (link)].