Ds qi1mc

Animal study was approved by the Institutional Animal Care and Use Committee of Tulane University (Protocol# 4040R, approved on 17 January 2011, valid through 16 January 2014). The Dcx-EGFP mice with a strain name of Tg (Dcx-EGFP) BJ224Gsat/Mmmh were obtained from the Mutant Mouse Regional Resource Center, University of Missouri, which were characterized previously [9 (link)]. Enhanced green fluorescence protein (eGFP) was expressed in Dcx-expressing cells in these mice. E12.5 and E13.5 mouse embryos were obtained through timed pregnancies. Images of mouse handplates were taken with an epifluorescence microscope (Nikon AZ100) equipped with a digital camera (Nikon DS-Qi1Mc) and NIS-Elements Basic 3.0 software (Nikon Instruments Inc., Melville, NY, USA). The handplates were dissected into different regions under the epifluorescence microscope, using VANNAS microdissecting spring scissors (Roboz Surgical, Gaithersburg, MD, USA). Approximately 16 handplates from 8 embryos of a single pregnant mouse each at E12.5 and E13.5 were collected and pooled.

BEAS-2B were cultured in four well chamber slides (Merck Millipore, Darmstadt, Germany) until approximately 60% confluency. Cells were treated with tryptase for 24 h followed by fixation in 2% paraformaldehyde for 20 minutes and thereafter washed in PBS. Samples were heated in antigen retrieval buffer (EnVision DAKO, Glostrup, Denmark) using DAKO PT LINK (Glostrup, Denmark) version 2.0.0. Samples with intracellular targets were permeabilized in PBS containing 0.1% Tween-20 for 10 minutes. Samples were immunostained with antibodies against KI67 (1:300) and CD151 (dilution 1:150) DAKO, Glostrup, Denmark and Sigma-Aldrich, St Louis, MO, USA, respectively) according to standard protocols. Antibodies against KI67 and CD151 were labeled with Alexa Fluor 488 diluted 1:200 (Invitrogen, Eugene, USA). Nuclei were counterstained with mounting medium ProLong Gold antifade reagent with DAPI (Invitrogen, Eugene, USA). Samples were viewed on Nikon eclipse 80i (Melville, NY, USA) combined with Nikon DS-QI1MC. Color intensity was quantified using ImageJ (Wisconsin, USA) by measuring the pixel intensity in relation to the threshold value which was determined based on untreated controls. The intensity measurement in these randomly chosen areas was then divided by the number of cells (based on nuclei DAPI staining) in each field of view.

Liver samples were excised from the right hepatic lobe, placed in a cartridge and fixed in 10% buffered formalin overnight and submitted to pathology core for hematoxylin and eosin staining. Slides were visualized under bright field with Nikon 80i microscope. Images were captured with a Nikon DS-Qi1MC camera and image analysis system (Nikon Instruments, Melville, NY).

HSCs, BMSC or whole bone marrow cells were plated overnight on coverslips coated with FBS for 30 min at RT then cells. Cells were fixed in 4% PFA for 15 min, washed and permeabilized with PBS/0.3%TritonX-100 for 3 min. Blocking was performed with 5% normal serum in permeabilization buffer for 1 hr at RT. Anti-Osx antibody (Abcam, #ab227820) was dissolved 1:500 in a buffer containing PBS/0.3%TritonX-100/1%BSA and incubated ON at 4°C. For fluorescent detection, coverslips were washed and incubated in AlexaFluor 488-conjugated anti-rabbit secondary antibody (1:1000) in PBS/0.3%TritonX-100/1%BSA for 1 hr at RT. Stained slides were washed and mounted using VECTASHIELD Mounting Medium containing DAPI (Vector Laboratories, CA USA). Fluorescent signals were captured by using a Nikon Eclipse 80i microscope and a Nikon DS-Qi1MC camera (Nikon, CO, USA).

To achieve optimal magnification and high resolution, the T. pallidum-platelet sample volume was limited to 4 μL per 1.0–1.2 mm thick glass slide with a 22 x 36 mm cover glass (Fisher Scientific Company, Ottawa, ON) gently pressed into place. Slides were viewed at 1000x oil magnification with a 100x oil pan-fluor objective lens set to 0.7 on a Nikon Eclipse 80i darkfield microscope with a Nikon DS-Qi1Mc digital camera with NIS-Elements imaging software (Nikon Canada Inc., Mississauga, ON). For video microscopy (high resolution real-time imaging), exposure times were set at 3–40 ms. For additional brightness, gain was increased to 9–16. Image clarity was directly related to the level of platelet activation and platelet microparticle (PMP) secretion. As activation and PMP secretion increased, additional light was scattered, resulting in a brighter field, and exposure times were further reduced to compensate. The quality of imaging samples with reduced light scattering was improved by increasing the exposure time to ≥ 60 ms and opting for still imaging rather than videos.

Bronchial and alveolar epithelial cells were cultured in chamber slides (Merck Millipore, Darmstadt, Germany) until 70–80% confluency and then treated with tryptase for 24 h. Samples were fixated in 2% paraformaldehyde for 20 min and washed in PBS. For the staining, samples were permeabilized in 0.1% Tween-20 for 10 min and immunostained with primary antibody cIAP-2/BIRC3 (Thermo Fisher Scientific, Waltham, MA, USA, PA551700) diluted 1:50, or Ki67 1:300 (M7240, Dako, Denmark), and thereafter incubated with Alexa Fluor 488 (Invitrogen, Eugene, OR, USA) diluted 1:200. ProLong Glass antifade with NucblueTM (Thermo Fisher Scientific, Waltham, MA, USA, P36983) was used for mounting and nuclei visualization. A Nikon eclipse 80i combined with Nikon DS-QI1MC was used to capture images and Image-J was used for data analysis. Data are shown as positive pixels per quantified cell nuclei. The magnification of the immunostaining was visualized using a Nikon A1+ confocal microscope with a 20× Plan Apo objective, NA 0.75 (Nikon Instruments Inc., Tokyo, Japan), and acquired using NIS-elements, version: 4.60.02 (Laboratory Imaging, Nikon, Tokyo, Japan). All image captures were taken with identical settings.