Coolsnap ez camera

Images were acquired with Zeiss Axio Imager A2 microscope with Cool Snap EZ camera (Visitron Systems) and MetaMorph Imaging software (MDS Analytical Technologies). For each pair of coverslips (treated vs. control) the same exposure time was taken. Per each experimental condition two coverslips were individually treated and processed. Images were captured from at least 3–5 visual fields (= cells) per coverslip and further analyzed using NIH ImageJ and OpenView software (Tsuriel et al., 2006 (link)). Upon appropriate background subtraction, immunoreactive puncta were counted along the 20 μm of proximal (≥10 μm and ≤50 μm distance from the cell body) or distal dendrite (≥50 μm distance from the cell body). The synaptic immunofluorescence intensities (IF) were assessed in a region of interest (ROI) set by the mask in the channel for synaptophysin (sph), which was used as synaptic marker. The mask was created semiautomatically using OpenView software.

Cover slips were coated with 100 µg/mL human fibrinogen and blocked with 1% BSA/PBS. After rinsing with Tyrodes-HEPES buffer, washed platelets (100 µL with 0.03×10⁶ platelets/µL) were activated with thrombin (0.01 U/mL) and immediately placed on the coated cover slips. At indicated time points, the cover slips were rinsed again and platelets were visualized with a Zeiss Axiovert 200 inverted microscope (100x/1.4 oil objective). Digital images were recorded using a CoolSNAP-EZ camera (Visitron) and analyzed off-line using Metavue software (Molecular Devises). Four different stages of platelet spreading were evaluated: stage 1 - roundish; stage 2 - filopodia only; stage 3 - filopodia and lamellipodia; stage 4 - fully spread.

Ibidi µ-Slide 8-well chambers were coated with 100 μg/ml human fibrinogen at 4°C overnight and blocked for at least 1h at RT with 1% BSA in sterile PBS. The wells were rinsed with Tyrode’s buffer and 300 μl washed platelets (250,000 cells/μl in Tyrode’s containing 2 mM Ca²⁺) were stimulated with thrombin (0.01 U/ml) and immediately added to the fibrinogen surface. Platelets were allowed to adhere and spread at 37°C for 10 min. The non-adhered platelets were washed away and the adhered cells were fixed by addition of 300 μl 4% PFA/PBS for 10 min at RT. Then, the wells were washed twice with 300 µl PBS. Platelets were visualized by differential interference contrast (DIC) microscopy with a Zeiss Axiovert 200 inverted microscope (100x/1.4 oil objective). Representative images were taken using a CoolSNAP-EZ camera (Visitron, Munich, Germany) and evaluated according to different platelet spreading stages with ImageJ (NIH) (42 (link)). Spreading stages were defined as follows: 1: round; no filopodia, no lamellipodia. 2: only filopodia. 3: lamellipodia; full spreading

For spreading on fibrinogen, coverslips were coated with 100 µg/ml human fibrinogen (Sigma) overnight at 4 °C and blocked with 1% BSA in PBS at 37 °C for 1 h. Slides were rinsed with Tyrode’s buffer before washed platelets were activated with 0.01 U/ml thrombin (Roche) and allowed to spread on the coated coverslips. Platelets were fixed with 4% PFA in PBS at different time points. Spread platelets were visualized with a Zeiss Axiovert 200 inverted microscope (100×/1.4 oil objective)^{43 (link)}. Digital images were recorded using a CoolSNAP-EZ camera (Visitron) and analyzed using ImageJ software.

Mesenteric arteries were exteriorized carefully and immobilized on a Petri dish. The injury of the arterioles was induced by application of a filter paper (3 mm² triangular) saturated with 20% FeCl₃. Thrombus formation of fluorescently labeled platelets (56F8 DyLight 488; anti-GPIX) was monitored up to 40 min or until occlusion (blood flow stopped for > 1 min). Images were recorded with a Zeiss Axiovert 200 inverted microscope (10x/0.60 objective) equipped with a CoolSNAP-EZ camera (Visitron).

Coverslips were coated with fibrinogen (100 μg/mL, F4883, Sigma-Aldrich) and blocked with 1% BSA/PBS. After washing with Tyrodes-HEPES buffer, washed platelets (3 × 10⁵ platelets/μL) were either unstimulated or activated with 0.01 U/mL thrombin (10602400001, Roche). At the respective time point the reaction was stopped by addition 4% PFA/PBS and images taken with a Zeiss Axiovert 200 inverted microscope (100x/0.60 objective) equipped with a CoolSNAP-EZ camera (Visitron) and analyzed off-line using ImageJ software. Four different stages of platelet spreading were evaluated: stage 1—roundish; stage 2—filopodia only; stage 3—filopodia and lamellipodia; stage 4—fully spread.

Image analysis was carried out using a Zeiss Axio Imager A2 fluorescent microscope (Zeiss, Jena, Germany) with Cool Snap EZ camera (Visitron System, Puchheim, Germany) and MetaMorph Imaging software (MDS Analytical Technologies, Ismaning, Germany). Up to 3 coverslips were treated individually and processed per group. For each coverslip, the same exposure time and intensity were taken among the different groups. After background subtraction, the fluorescence intensity of the immunosignal was measured along dendrites right after the first branching point using ImageJ software. The synaptic immunofluorescence intensities of pan-AKT and phospho-AKT were assessed in a region of 400 nm × 400 nm square set by the mask generated based on synaptic marker Shank3. The Shank3 mask was created semi-automatically using OpenView software [34 (link)].