Light microscopic images of primo-vascular system. (A) Primo vessel (PV) and primo nodes (PN) on surface of small intestine. Free movable PVS is found as part of larger network on/between internal organs. Note transparent PV and PN compared with scarlet blood vessels (BV). (B) PV inside lymphatic vessel (LV) stained with Alcian blue. PV inside LV was clearly visible as a blue line 15 min after injection of 0.1% Alcian blue into lumbar lymph node. Scale bars indicate 200 μm.|@|~(^,^)~|@|Binding affinity of α-rPVS-mAb to the proteins of PVS, and images of Coomassie blue (CB)-stained SDS gel and WB. (A) Binding affinity of each α-rPVS-mAb to the protein of PVS by ELISA. The isolated proteins of rat PVS was coated onto a microplate and processed with supernatant of each hybridoma cell. Binding affinity was shown as OD₄₀₅ value after color reaction of immunogen-antibody. α-rPVS-m3-4 showed the strongest affinity to proteins of PVS. (B) CB stained SDS polyacrylamide gel. Lane M and P lane were electrophoresed with a size marker of molecular weight and isolated total proteins of PVS. (C) Corresponding WB of lane P in (B). Lane 1, 2, and 3 was probed with α-rPVS-m3-2, α-rPVS-m1-1, and α-rPVS-m4-6, respectively. Note WB signal strength of each mAb to proteins of PVS from Table 1.|@|~(^,^)~|@|Immunofluorescence microscopic (IF) images of blood vessel (BV), lymphatic vessel (LV), and primo vessel (PV) as negative controls. Longitudinally sectioned BV (A-C) and LV (D-F) were probed with α-rPVS-m3-2. DNA and signal of immune reaction on sections were detected by DAPI (A, D) and 2^nd Ab of goat anti-mouse IgG-Dylight 488 (B, E), respectively. Images of IF in B and E represent those probed with all the α-rPVS-mAb, indicating that the generated mAbs did not bind to BV and LV in rat. Section of PV (G-I) was probed with α-rPVS-m1-1 and then treated with DAPI (G) and 2^nd Ab (H) to detect DNA and signal of immune reaction. α-rPVS-m1-1 showed no signal in IF of PVS although it exhibited positive signals both ELISA and WB as shown in Table 1. Scale bars indicate 100 μm.|@|~(^,^)~|@|Immunofluorescence microscopic (IF) images of PV and PN probed with α-PVS-m3-2. Cross (A-C) and longitudinal section (D-F) of PV and PN (G-H) were probed with α-rPVS-m3-2 and localization of immunogen was detected by 2^nd Ab (goat anti-mouse IgG)-Dylight 488 (B, E, H). α-rPVS-m3-2 was predominantly localized on cell membrane and extra-cellular matrix of PV (arrow in B, E) and PN (arrow in H). Note that there were two different types of sinus in PN; insets by open and white triangle. Sinus by open triangle was not stained but sinus by white triangle was stained at its outer surface. Scale bars indicate 100 μm.|@|~(^,^)~|@|Immunofluorescence microscopic (IF) images of PV and PN probed with α-PVS-m3-4. Cross (A-C) and longitudinal section (D-F) of PV and PN (G-H) were probed with α-rPVS-m3-4 and localization of immunogen was detected by 2^nd Ab-Dylight 488 (B, E, H). α-rPVS-m3-4 was bound to on cell membrane and extra-cellular matrix of PV and PN (arrow in B, E, H). It was also bound to surface of sub-ducts of PV and PN (arrowhead in B, H). Scale bars indicate 100 μm.|@|~(^,^)~|@|Immunofluorescence microscopic (IF) images of PV inside lymphatic vessel (LV). Cross (A-C) and longitudinal section (D-F) of LV were probed with α-rPVS-m3-2, and then processed to visualize reaction of immunogen-mAb by 2^nd Ab-Dylight 488 (B, E). The signal appeared only on PV but not on LV, indicating that α-rPVS-mAb has specificity to PV of rat in IF. Scale bars indicate 100 μm.

2020;13:110~115 https://doi.org/10.1016/j.jams.2020.05.001