sheep anti human cd31 pecam 1  (R&D Systems)

Journal: bioRxiv

Article Title: Paracrine Factor Local Gradient-Generating System for Engineering Perfusable Vascularized Hepatocyte Tissues with Perfusion-Induced Proliferation

doi: 10.1101/2025.11.10.687539

Figure Lengend Snippet: A: Schematic illustrations of PFLG generated by hLF cryogel, inducing microvessel penetration into hepatocyte tissue. B: Fluorescent projection images of vascularized hepatocyte tissue on day 5. Blue: cell nuclei (Hoechst 33342), green: F-actin (Alexa Fluor 488-phalloidin), red: HUVECs (CD31). Arrowheads indicate microvessels penetrating hepatocyte tissue. Asterisks in the magnified image indicate dead cells in hepatocyte tissue. Scale bars, 200 µm (upper panel); 50 µm (lower panel, magnified images). C: Quantitative analysis of vessel area ratio in fluorescent projection images of vascularized hepatocyte tissues on day 5. D: Quantitative analysis of the number of penetrating vessels on day 5. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (One-way ANOVA followed by Tukey’s HSD test).

Article Snippet: Samples were then incubated overnight at 4°C with the following primary antibodies: sheep anti-PECAM-1 (CD31) (AF806, R&D Systems, Minneapolis, MN, USA) to label HUVECs; mouse anti-multidrug resistance-associated protein 2 (MRP2) (NBP1-42349, Novus Biologicals, Centennial, CO, USA) to visualize bile canaliculi formed by rHeps; and rabbit anti-Ki67 (ab16667, Abcam, Cambridge, UK) to identify proliferating cells.

Techniques: Generated

Journal: bioRxiv

Article Title: Paracrine Factor Local Gradient-Generating System for Engineering Perfusable Vascularized Hepatocyte Tissues with Perfusion-Induced Proliferation

doi: 10.1101/2025.11.10.687539

Figure Lengend Snippet: A: Immunofluorescence confocal images of vascularized hepatocyte tissue (rHep:HUVEC = 9:1) on day 5. Blue: cell nuclei (Hoechst 33342), green: F-actin (Alexa Fluor 488-phalloidin), red: HUVECs (CD31). B: Cross-sectional images of vascularized hepatocyte tissue. Arrowheads indicate microvessels penetrating the hepatocyte tissue. The asterisk in the magnified XZ section highlights hepatic sinusoid-like structures, where hepatocytes are in close contact with microvessels. Scale bar, 50 µm. C: Schematic illustrations of hepatic sinusoids (H: hepatocytes, D: space of Disse, SEC: sinusoid endothelial cells, S: hepatic sinusoid).

Article Snippet: Samples were then incubated overnight at 4°C with the following primary antibodies: sheep anti-PECAM-1 (CD31) (AF806, R&D Systems, Minneapolis, MN, USA) to label HUVECs; mouse anti-multidrug resistance-associated protein 2 (MRP2) (NBP1-42349, Novus Biologicals, Centennial, CO, USA) to visualize bile canaliculi formed by rHeps; and rabbit anti-Ki67 (ab16667, Abcam, Cambridge, UK) to identify proliferating cells.

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: Paracrine Factor Local Gradient-Generating System for Engineering Perfusable Vascularized Hepatocyte Tissues with Perfusion-Induced Proliferation

doi: 10.1101/2025.11.10.687539

Figure Lengend Snippet: A: Immunofluorescence projection images of bile canalicular formation in vascularized hepatocyte tissue. Projection images were constructed from Z-stack confocal images. Blue: cell nuclei (Hoechst 33342), green: F-actin (Alexa Fluor 488-phalloidin), red: HUVECs (CD31), pink: bile canaliculi (MRP2). Arrowheads indicate MRP2-positive bile canaliculi. B: Excretion of CellTracker Red CMTPX by polarized hepatocytes in vascularized hepatocyte tissue (red: CMTPX; green: GFP-rHep). Arrowheads indicate CMTPX excreted into bile canaliculi. C: Cross-sectional image showing CMTPX excretion into bile canaliculi in vascularized hepatocyte tissue (red: CMTPX; green: GFP-rHep). Arrowheads indicate CMTPX in bile canaliculi. Scale bars, 50 µm. D: Quantitative analysis of MRP2-positive area ratio in hepatocyte tissue. E: Quantification of albumin secretion from hepatocyte tissues. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (One-way ANOVA followed by Tukey’s HSD test).

Article Snippet: Samples were then incubated overnight at 4°C with the following primary antibodies: sheep anti-PECAM-1 (CD31) (AF806, R&D Systems, Minneapolis, MN, USA) to label HUVECs; mouse anti-multidrug resistance-associated protein 2 (MRP2) (NBP1-42349, Novus Biologicals, Centennial, CO, USA) to visualize bile canaliculi formed by rHeps; and rabbit anti-Ki67 (ab16667, Abcam, Cambridge, UK) to identify proliferating cells.

Techniques: Immunofluorescence, Construct

Journal: bioRxiv

Article Title: Paracrine Factor Local Gradient-Generating System for Engineering Perfusable Vascularized Hepatocyte Tissues with Perfusion-Induced Proliferation

doi: 10.1101/2025.11.10.687539

Figure Lengend Snippet: A: Time-lapse images of fluorescently labeled microbeads (diameter: 1 µm) perfused through vascularized hepatocyte tissue. Arrowheads indicate a perfused microbead, and arrows indicate the flow direction within the microvessel. Scale bar, 50 µm. B: Time-lapse and projection images showing perfusion of 70 kDa FITC-dextran solution through vascularized hepatocyte tissue. Green: FITC-dextran and F-actin (Alexa Fluor 488-phallidin), red: microvessels (CD31). Scale bars, 200 µm (upper panel); 100 µm (lower panel, magnified images). C: Quantitative analysis of cumulative FITC-dextran fluorescence in regions H1–H5 and V1–V5 shown in the right image. Boxes in the projection image indicate regions of interest for analyzing cumulative FITC-dextran fluorescence during the perfusion process (H1–H5: hepatocyte tissue regions; V1–V5: hydrogel regions)

Article Snippet: Samples were then incubated overnight at 4°C with the following primary antibodies: sheep anti-PECAM-1 (CD31) (AF806, R&D Systems, Minneapolis, MN, USA) to label HUVECs; mouse anti-multidrug resistance-associated protein 2 (MRP2) (NBP1-42349, Novus Biologicals, Centennial, CO, USA) to visualize bile canaliculi formed by rHeps; and rabbit anti-Ki67 (ab16667, Abcam, Cambridge, UK) to identify proliferating cells.

Techniques: Labeling, Fluorescence

Journal: bioRxiv

Article Title: Paracrine Factor Local Gradient-Generating System for Engineering Perfusable Vascularized Hepatocyte Tissues with Perfusion-Induced Proliferation

doi: 10.1101/2025.11.10.687539

Figure Lengend Snippet: A: Schematic illustrations of static and perfusion culture conditions. Cells were cultured with hLF cryogels (hLF(+)) until day 3. From day 3 to day 7, some cells were cultured without hLF cryogels (hLF(−)) under either static (0 mmH 2 O) or perfusion (1 or 2 mmH 2 O) conditions. B: Immunofluorescence projection images of vascularized hepatocyte tissue under static and perfusion culture. Projection images were constructed from Z-stack confocal images. Blue: cell nuclei (Hoechst 33342), green: F-actin (Alexa Fluor 488-phalloidin), red: HUVECs (CD31), yellow: proliferating cell nuclei (Ki67). Arrowheads indicate Ki67-positive proliferating hepatocytes. Asterisks indicate bile canaliculi-like structures, showing double-layered F-actin between hepatocytes. Scale bars, 50 µm. C: Representative immunofluorescence images showing Ki67-positive rHeps undergoing mitosis. Arrowheads indicate both interphase nuclei and mitotic chromosomal dynamics. Blue: cell nuclei (Hoechst 33342), green: F-actin (Alexa Fluor 488-phalloidin), red: HUVECs (CD31), yellow: proliferating cell nuclei (Ki67). D: Quantification of the ratio of Ki67-positive rHeps. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (One-way ANOVA followed by Tukey’s HSD test).

Article Snippet: Samples were then incubated overnight at 4°C with the following primary antibodies: sheep anti-PECAM-1 (CD31) (AF806, R&D Systems, Minneapolis, MN, USA) to label HUVECs; mouse anti-multidrug resistance-associated protein 2 (MRP2) (NBP1-42349, Novus Biologicals, Centennial, CO, USA) to visualize bile canaliculi formed by rHeps; and rabbit anti-Ki67 (ab16667, Abcam, Cambridge, UK) to identify proliferating cells.

Techniques: Cell Culture, Immunofluorescence, Construct