Polyclonal rabbit anti human vwf

Pulmonary vascular remodeling was evaluated as previously described [36 (link)]. Briefly, lung sections were stained with polyclonal rabbit anti-human vWF (Dako), mouse anti α-smooth muscle actin (SMA,1:500; Sigma, St Louis, MO) and 4′,6-diamidino-2-phenylindole (DAPI, Vector Biolabs, Burlingame, CA). The percentage of peripheral pulmonary vessels (< 50 μm in diameter) stained with α-SMA > 50% of the circumference was determined from ten random images on each lung section and all analyses were performed by a blinded observer.

Polystyrene plates were coated with 100 µL of collagen type III (0.3 µg/mL) or a 2% (w/v) solution of BSA diluted in PBS for two hours at 37°C. After washing twice with PBS to remove unbound protein, residual binding sites were blocked by adding 5 mg/mL denatured BSA overnight at 4°C. After washing 3× with 50 mM Tris HCl, 150 mM NaCl, and 0.05% (v/v), TBS-T, pH 7.4, increasing concentrations of recombinant Simplagrin (ranging from 0.0015 to 1.5 µM) were added to the wells and incubated at 37°C for one hour. Wells were washed again and incubated with 3 nM of vWF factor VIII free (Haematologic Technologies Inc.) in TBS-T supplemented with 2% (w/v) BSA. After one hour at 37°C, wells were washed 3× with TBS-T, and a polyclonal rabbit anti-human vWF (DakoCytomation) was added (1∶500 in TBS-T) and incubated for one hour at 37°C. After three washes with TBS-T, an alkaline phosphatase conjugate anti-rabbit IgG (whole molecule; Sigma) was added (1∶10000) and incubated at 37°C for 45 minutes. Before adding the stabilized p-nitrophenyl phosphate liquid substrate (Sigma), wells were washed 6× with TBS-T. After 30 minutes of substrate conversion, the reaction was stopped with 3 N NaOH and absorbance read at 405 nm using a Thermomax microplate reader (Molecular Devices). Wells coated only with BSA were used as assay blanks. All experiments were performed in triplicate.

Localization of VWF within the injured kidney was investigated by immunofluorescence double staining using confocal laser scanning microscopy (Zeiss LSM 710, Zeiss, Germany). After cutting, antigen retrieval and blocking, formalin-fixed paraffin sections were incubated with polyclonal rabbit anti-human VWF (Dako, Copenhagen, Denmark) and monoclonal rat anti-mouse CD42b (GPIb, emfret Analytics, Eibelstadt, Germany), as well as the secondary antibodies Alexa555-conjugated goat anti-rat (Invitrogen, Eugene, Oregon, USA) and FITC-conjugated goat anti-rabbit (BD Pharmingen, USA), as described previously^{6 ,32 (link)}.