It has also been Bortezomib demonstrated by other investigators that FH was not detected in the ��-granules of platelets using either fluorescent confocal microscopy or electron microscopy. In contrast, in early 2014, Rayes, et al reported FH and VWF colocalization in HUVEC WPBs by using two secondary antibodies with wavelengths that have some spectral overlap. In the report by Rayes, et al, the high intensity of VWF fluorescence in WPBs may have caused a ��bleed through�� into the 488 nm channel and caused the authors to identify mistakenly this latter fluorescence as ��FH�� co-localizing with VWF in WPBs. In our current experiments, we used non-overlapping spectral secondary detection antibody pairs, and did not detect FH in HUVEC WPBs. Specifically, the conclusion drawn by Rayes, et al that FH is present along with VWF in HUVEC WPBs was based on two types of experiments done by their group: detection of FH and VWF in HUVEC WPBs using fluorescent microscopy; and measurement of VWF and FH levels released into HUVEC supernatant with and without heme exposure. In their fluorescent microscopy experiments, FH was detected using 3 biotinylated monoclonal antibodies against FH plus secondary streptavidin-FITC. VWF was detected with a polyclonal rabbit antibody against VWF plus secondary goat anti-rabbit IgG-Alexa Fluor -555. The emission spectrum for FITC ranges from 486 to 656 nm and the emission spectrum range for AF-555 is 540 to 705 nm. There is a spectral overlap from 540 nm through 655 nm that would allow the VWF-AF-555 emission signal to be detected in the FH-FITC channel. Because FH is located throughout the HUVEC cytoplasm, the single-channel images of FH detection shown by Rayes, et al show a green cytoplasmic background plus green WPBs from the AF-555 signal ��bleed through��. Their merged images of FH and VWF have green FH cytoplasmic staining plus yellow WPBs indicating overlapping detection in both channels. In their paper, the authors did not question why FH could have 2 separate cellular locations. The Method section in the Rayes, et al article did not provide details about instrumentation, image acquisition, and number of experiments. In contrast, in this current manuscript we detected VWF using rabbit anti-human VWF antibody plus chicken anti-rabbit IgG-AF-488-secondary antibody, and detected FH with either a combination of two mouse anti-human FH monoclonal antibodies ] plus goat anti-mouse F 2-IgG-AF 647-secondary antibody or with a polyclonal goat anti-human FH antibody plus donkey anti-goat IgG-AF 647-secondary antibody. The emission spectrum for AF-488 is the same as for FITC 9described in the proceeding paragraph). The excitation peak for AF-647 is 650 and the emission peak is at 668 nm. The emission spectral range for AF-647 is from 629 to 800 nm, and there are no overlapping spectral regions for AF-647 and AF-488. SCH772984 Furthermore, our previous 2013 PLoS ONE article contains a figure demonstrating the lack of signal ��bleedthrough�� in our fluorescent microscope system. In these previously published experiments, VWF was detected with AF-488 in HUVEC WPBs and cytoplasmic complement proteins were detected with AF-594-labeled secondary antibodies. We measured the signal intensities along lines dissecting WPBs in the 488-nm channel and intensities at identical locations detected in the 594-nm channel in order to prove the lack of signal ��bleed-through��. In the Rayes, et al article, levels of FH and VWF were measured in untreated HUVEC supernatant or HUVECs exposed to 100 ��Mheme for 30 min. Their VWF and FH levels increased from ~35 to 80 ng/ml and from ~0.5 to 1.7 ng/ml, respectively.