Fluoromount

After FeCl₃-induced injury, the occluded carotid artery was removed and embedded in paraffin. Paraffin sections were stained with biglycan-specific antibody (biglycan polyclonal rabbit antibody, abcam, Cambridge, United Kingdom, # 49701, 1:100) following the streptavidin–biotin immuno-peroxidase method (Dako, Glostrup, Denmark). Sample slices of healthy carotid arteries were prepared from paraffin-embedded samples, dewaxed and dehydrated, blocked (5% goat serum, 0.1% BSA and 0.3% triton (Sigma-Aldrich, Burlington, MA, USA, # T8787-100)), and incubated with antibodies against collagen I (rabbit anti-mouse collagen I antibody, Thermo Fisher Scientific Inc., Waltham, MA, USA, # PA1-85319) and collagen IV (rabbit polyclonal collagen IV antibody, abcam, Cambridge, United Kingdom, # ab6586). Then, secondary antibody incubation was performed for 1 h (Alexa Fluor™ 555 goat anti-rabbit IgG (Thermo Fisher Scientific Inc., Waltham, MA, USA # A21428)), and cell nuclei were stained with DAPI (Roche Diagnostics, Mannheim, Germany, final concentration 1.6 µg/mL) and inundated with FluoromountTM (Sigma-Aldrich, Burlington, MA, USA). The documentation was done after drying at 4 °C overnight with microscope Axio Observer.D1 (Carl Zeiss Microscopy GmbH, Oberkochem, Germany, AxioCam MRm, objective LD Plan-Neofluar 40× 0.6 Korr. Ph2 M27).

A protocol based on the Wachstein/Meisel lead phosphate method was used [8 (link),11 (link),44 (link),45 ]. The sections were washed twice with 50 mM Tris-maleate buffer pH 7.4 and pre-incubated for 30 min at RT with 50 mM Tris-maleate buffer pH 7.4 containing 2 mM MgCl₂ and 0.25 mM sucrose. The enzymatic reaction was carried out by incubating tissue sections for 1 h at 37 °C with 50 mM Tris-maleate buffer pH 7.4 supplemented with 0.25 mM sucrose, 2 mM MgCl₂, 5 mM MnCl₂, 3 % Dextran, 2 mM Pb(NO₃)₂, and 2 mM CaCl₂. All experiments were performed in the presence of 2.5 mM levamisole, as an inhibitor of alkaline phosphatase (AP) activity, and in the presence of 1 mM AMP, ADP, ATP, or TPP as a substrate. TPP is a false substrate, which can be cleaved by the pyrophosphatase activity of E-NPPs. Control assays were performed in the absence of nucleotide. For E-NTPDase inhibition experiments, 1 mM POM 1 was added to pre-incubation and enzymatic reaction buffers. For CD73 inhibition experiments, 1 mM α, β-meADP was added to pre-incubation and enzymatic reaction buffers. The reaction was revealed by incubation with 1% (NH₄)₂S (v/v) for exactly 1 min. Nuclei were counterstained with haematoxylin. Samples were mounted with aqueous mounting medium (FluoromountTM, Sigma-Aldrich), observed under a light Nikon Eclipse E200 microscope, and photographed under a light Leica DMD 108 microscope.

Experiments were performed as described previously [21 (link),29 (link)]. Coverslips (24 × 60 mm) were either coated with 100 µg/mL fibrinogen or 200 μg/mL type I collagen at a defined area (10 × 10 mm) and incubated overnight at 4 °C. Afterwards the coverslips were blocked with 1% BSA for 60 min. Then, 2 × 10⁴ isolated platelets were resuspended in 70 μL Tyrode′s buffer (pH 7.35) supplemented with CaCl₂ (2 mM), applicated to the prepared coverslips and incubated at room temperature for indicated time points. Non-adherent platelets were carefully removed by rinsing 2× with PBS. The preparation was fixed by 4% phosphate buffered formaldehyde at 4 °C for 10 min, subsequently rinsed again carefully with PBS and inundated with FluoromountTM (Sigma-Aldrich, St. Louis, MO, USA). Platelet adhesion was documented after drying at 4 °C overnight using the microscope Axio Observer.D1 (Carl Zeiss Microscopy GmbH, Oberkochem, Germany, AxioCam MRm, objective LD Plan-Neofluar 40x/0.6 Korr Ph2 M27). The images were analyzed ImageJ-win64 (ImageJ 1.52, freeware by National Institutes of Health, USA) and ZEN 2.6 (ZEN 2.6 blue edition, Carl Zeiss Microscopy GmbH, Oberkochem, Germany).

For visualization, two drops of peripheral blood were added to 50 µL heparin (20 U/mL). Then one drop was spread on a cover slip and the resulting blood smears were stained with DAPI (Roche Diagnostics, Mannheim, Germany final concentration 1.6 µg/mL) to stain nuclei of leukocytes. Images were taken with the Zeiss LSM 510-META Confocal laser scanning microscope (Carl Zeiss, Oberkochem, Germany).
Sample slices were prepared from paraffin embedded spleen and femur samples, dewaxed and dehydrated, stained with DAPI (Roche Diagnostics, Mannheim, Germany, final concentration 1.6 µg/mL) and inundated with FluoromountTM (Sigma-Aldrich, St. Louis, MO, USA). The documentation was done after drying at 4 °C overnight with microscope Axio Observer.D1 (Carl Zeiss Microscopy GmbH, Oberkochem, Germany, AxioCam MRm, objective LD Plan-Neofluar 40x 0.6 Korr Ph2 M27) and the resulting pictures were analyzed using ImageJ-win64 (ImageJ 1.52, freeware by National Institutes of Health, USA) and ZEN 2.6 (ZEN 2.6 blue edition, Carl Zeiss Microscopy GmbH, Oberkochem, Germany).

Neurons on glass coverslips were fixed for 5 min in 4% PFA/4% sucrose at RT, permeabilized with PBS^+/+ (D8662, Sigma, supplemented with 1 mM MgCl2 and 0.1 mM CaCl₂))/Triton-0.1%, blocked with PBS^+/+/5% BSA and labeled with primary antibodies in PBS^+/+ (D8662, Sigma) and 5% BSA for 2 h and secondary antibodies for 45 min. PBS^+/+ washes were performed after each antibody incubation. Coverslips were mounted on glass slides in Fluoromount^TM (F4680, Sigma). Images were captured using Zeiss LSM-700 system with a Plan-Apochromat 63 × /NA 1.40 oil DIC, using Zen 2010 software.
Primary antibodies used: mouse anti-GB1 (ab55051, Abcam), chicken anti-map2 (ab5392, Abcam), mouse anti-c-myc 9E10 (11667149001, Roche), mouse anti-piccolo (142111, Synaptic System), rabbit anti-KLC1 (ab187179, Abcam), rabbit anti-GFP (ab290, Abcam), mouse anti-flag M2 (F1804, Sigma), mouse anti-PSD-95 (ab2723, Abcam). Secondary antibodies used: Alexa Fluor® 647 donkey anti-chicken IgY (Millipore), Alexa Fluor® 555 donkey anti-mouse IgG (Life Technologies), Alexa Fluor® 488 donkey anti-rabbit IgG (Life Technologies), Alexa Fluor® 647 donkey anti-mouse IgG (Invitrogen), and Alexa Fluor® 568 donkey anti-rabbit IgG (Invitrogen).

The acrosomal exocytosis evaluation was performed as previously described [36 ]. Slides were washed for 5 min twice in PBS and 50 µL of 15 µg/mL of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) (L7381, Merck) and 6.5 µg/mL Hoechst 33,342 in PBS were added and covered with a 24 × 24 cover slide. The slides were then incubated for 30 min at room temperature in a humid box. Subsequently, the slides were washed in distilled water for 10 min and mounted with Fluoromount^TM aqueous mounting medium (Sigma-Aldrich, St. Louis, MO, USA), sealed with nail polish and examined in a fluorescence microscope Nikon Optiphot-2 (Nikon, Tokyo, Japan). At least 200 spermatozoa from two slides per sample were analysed by blind counting using codified slides. Only completely reacted acrosomes were counted as reacted and spermatozoa showing partial staining in the acrosome were counted as unreacted.

After treatment with WABI, MM1R cells were fixed in 4% (v/v) formaldehyde. Cells were stained with rhodamine–phalloidin (Molecular Probes, Thermo Fisher Scientific Inc., Waltham, MA, USA) to visualize the cellular morphology, Hoechst 33,258 (Sigma-Aldrich, St. Louis, MO, USA) to visualize the nucleus and streptavidin-Alexa Fluor555 (Molecular Probes, Thermo Scientific, MA, USA) to visualize biotinylated WA. To determine WABI colocalization with specific proteins (BTK), WABI-treated MM1R cells were immunostained with primary (anti-rabbit BTK, Cell Signaling Technology, mAb#8547) and corresponding labeled secondary antibodies (anti-rabbit Alexa Fluor-488, Molecular Probes, Thermo Scientific, MA, USA). Cells were then placed on glass slides and mounted with coverslips using Fluoromount^TM (Sigma-Aldrich, St. Louis, MO, US)Confocal imaging was carried out by a laser-scanning microscope equipped with a Plan-Apochromat 63X/1.40 Oil DIC objective lens and excitation wavelengths 405, 488, 561 and 640 nm (Zeiss LSM 800, Carl Zeiss, Germany). At least 20 different microscopic fields were analyzed for each sample using ZEN imaging software (Carl Zeiss). ZEN lite^TM (Carl Zeiss, Germany) was used to perform image reconstruction and presentation.