Horseradish peroxidase conjugated polyclonal rabbit anti human vwf

Electrophoresis was performed as per previously published methodology [16 (link)]. Briefly, samples were run on a gel using high gelling temperature SeaKem agarose (Lonza, Basel, Switzerland) and transferred to a PVDF membrane (Merck KGaA, Darmstadt, Germany). Membranes were blocked using 5% skim milk in TBS/Tween and probed with horseradish peroxidase conjugated polyclonal rabbit anti-human vWF (1:1000) (DAKO, Glostrup, Denmark). Membranes were washed and developed with enhanced chemiluminescence (ECL; Amersham Bioscience, NJ, USA), and band density visualized with the ImageQuant LAS 4000 (GE Healthcare, NJ, USA). Protein expression was quantified using ImageJ (National Institute of Health) and data normalised to baseline measurements.

Electrophoresis was performed as previously described.³⁹, ⁴⁰ Briefly, PPP samples were run through a high gelling temperature SeaKem agarose gel (Lonza, Basel, Switzerland) and transferred to an Immobilon‐P transfer membrane (Merck KGaA, Darmstadt, Germany). Membranes were blocked using 5% skim milk in Tris Buffered Saline with Tween (Sigma Aldrich, St. Louis, MO, USA) and probed with horseradish peroxidase‐conjugated polyclonal rabbit anti‐human vWF (1:1000) (DAKO, Glostrup, Denmark). Membranes were washed and developed with enhanced chemiluminescence (Clarity ECL Western Blotting Substrate, Bio‐Rad Laboratories, Inc, Hercules, CA, USA), and vWF multimer band density visualized with an ImageQuant LAS 4000 (GE Healthcare, Chicago, IL, USA). The densities of HMW vWF multimers were quantified using standard software (Image J, version 1.8.0_112, National Institute of Health, Bethesda, MD, USA). The HMW vWF multimer density of each condition sample was normalized to the static (0 s⁻¹) samples as a baseline and expressed as mean fold change ± standard deviation.