Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver.

doi: 10.1152/ajpgi.00139.2023

Figure Lengend Snippet: Figure 2. Three-dimensional (3-D) visualization of lymphatic vessels in the portal tract of normal mouse and human livers. A, left: reconstructed 3-D image of the portal tract in the normal mouse liver. Imaging condition: z-step size ¼ 0.4 μm, image volume [x] [y] [z] ¼ 1,290 μm 1,290 μm 354 μm. Liver tissue was cleared and stained with LYVE1 (red, lymphatic vessel), ASMA (white, portal vein), and CK19 (green, bile duct) antibodies. Right: 3-D volume-rendering image of lymphatic vessels and bile ducts using Imaris software with an enlarged image. Scale bars: 100 μm. B: reconstructed 3-D image of the portal tract in normal human liver tissue. Imaging condition: z-step size ¼ 0.4 μm, image volume [x] [y] [z] ¼ 1,002 μm 1,168 μm 228 μm. Liver tissue was cleared and stained with PDPN (red, lymphatic vessel) and ASMA (white, portal vein) antibodies. Scale bar: 100 μm. C, left: recon- structed 3-D image of the portal tract in normal human liver tissue. Imaging condition: z-step size ¼ 0.4 μm, image volume [x] [y] [z] ¼ 1,193 μm 1,193 μm 233 μm. Liver tissue was cleared and stained with PDPN (red, lymphatic vessel) and CK19 (green, bile duct) antibodies. Right, top: enlarged reconstructed 3-D image. Right, bottom: 3-D volume-rendering image. Scale bars: 100 μm.

Article Snippet: The liver slides were washed with PBS and incubated in blocking buffer (PBS containing 5% donkey serum and 0.3% triton X-100) at room temperature for 1 h. After blocking was completed, the liver slides were incubated with goat anti-mouse LYVE1 (1:200, AF2125, R&D Systems, Minneapolis, MN) (17), sheep anti-rat LYVE1 (1:100, AF7939, R&D Systems) (18), and rabbit anti-COL-I (1:100, ab292, Abcam) (19) primary antibodies at 4 C overnight, followed by washing with PBS for 5 min for 3 times and incubation with secondary antibodies including donkey anti-goat Alexa 647 (1:300, A32849, Invitrogen), donkey anti-sheep Alexa 647 (1:300, A21448, Invitrogen), and donkey anti-rabbit Alexa 488 at room temperature for 30 min. For immunostaining of PDPN in human liver slides, we amplified fluorescence signal using the avidin-biotin amplification method (SP-2001, Vector Laboratories, Newark, CA).

Techniques: Imaging, Staining, Software

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver.

doi: 10.1152/ajpgi.00139.2023

Figure Lengend Snippet: Figure 3. Lymphatic vessels in the capsular area of normal mouse, rat, and human livers. A: comparison of the capsular areas (thickness) among normal human (n ¼ 4), mouse (n ¼ 7), and rat livers (n ¼ 7) stained with Sirius Red. Scale bars: 100 μm. B: two-dimensional (2-D) conventional histology images of the capsular areas (paraffin section, 7-lm thickness) from normal human, mouse, and rat livers stained with PDPN or LYVE1 (red, lymphatic vessel) and Col-I (green, collagenous capsular area), respectively. Yellow arrows indicate lymphatic vessels inside the Col-I positive capsular area. 2-D images were merged with differential interference contrast (DIC) images. Scale bars: 100 μm. C: surface of human liver tissue (top) covered by thin collagenous cap- sule (Glisson’s capsule, yellow arrowhead, bottom) was isolated from normal human liver tissue to visualize capsular lymphatic vessels. D, left: recon- structed 3-D image of lymphatic vessels (PDPN, red) in the capsular area of normal human liver tissue. Imaging condition: z-step size ¼ 0.3 μm, image volume [x] [y] [z] ¼ 1,551 μm 1,551 μm 145 μm. Scale bars: 100 μm. Right: 3-D volume-rendering image. Scale bars: 100 μm.

Article Snippet: The liver slides were washed with PBS and incubated in blocking buffer (PBS containing 5% donkey serum and 0.3% triton X-100) at room temperature for 1 h. After blocking was completed, the liver slides were incubated with goat anti-mouse LYVE1 (1:200, AF2125, R&D Systems, Minneapolis, MN) (17), sheep anti-rat LYVE1 (1:100, AF7939, R&D Systems) (18), and rabbit anti-COL-I (1:100, ab292, Abcam) (19) primary antibodies at 4 C overnight, followed by washing with PBS for 5 min for 3 times and incubation with secondary antibodies including donkey anti-goat Alexa 647 (1:300, A32849, Invitrogen), donkey anti-sheep Alexa 647 (1:300, A21448, Invitrogen), and donkey anti-rabbit Alexa 488 at room temperature for 30 min. For immunostaining of PDPN in human liver slides, we amplified fluorescence signal using the avidin-biotin amplification method (SP-2001, Vector Laboratories, Newark, CA).

Techniques: Comparison, Staining, Isolation, Imaging

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver.

doi: 10.1152/ajpgi.00139.2023

Figure Lengend Snippet: Figure 4. Spatial relationship between lymphatic vessels and bile ducts in biliary cirrhotic livers. A: reconstructed three-dimensional (3-D) image of the portal tract in the liver from a sham-operated mouse (EC-GFP reporter mouse). Imaging condition: z-step size ¼ 0.4 μm, image volume [x] [y] [z] ¼ 1,025 μm 1,161 μm 211 μm. Liver tissue was cleared and stained with LYVE1 (red, lymphatic vessel: LV) and CK19 (white, bile duct: BD). Scale bar: 100 μm. B: reconstructed 3-D image of the portal tract in the liver from a 4-wk BDL mouse (EC-GFP reporter mouse) with a representative single optical sec- tion in the right. Imaging condition: z-step size ¼ 0.4 μm, image volume [x] [y] [z] ¼ 862 μm 939 μm 220 μm. Liver tissue was cleared and stained with LYVE1 (red, lymphatic vessel) and CK19 (white, bile duct). LV, lymphatic vessel; BD, bile duct; PV, portal vein; HA, hepatic artery; PBP: peribili- ary plexus. Scale bars: 100 μm. C: 3-D volume-rendering image of lymphatic vessels and bile ducts in the portal tract with enlarged images (D–F). Scale bar: 100 μm. BDL, bile duct ligation; EC-GFP, Endothelial-specific green fluorescent protein mice; LV, lymphatic vessel.

Article Snippet: The liver slides were washed with PBS and incubated in blocking buffer (PBS containing 5% donkey serum and 0.3% triton X-100) at room temperature for 1 h. After blocking was completed, the liver slides were incubated with goat anti-mouse LYVE1 (1:200, AF2125, R&D Systems, Minneapolis, MN) (17), sheep anti-rat LYVE1 (1:100, AF7939, R&D Systems) (18), and rabbit anti-COL-I (1:100, ab292, Abcam) (19) primary antibodies at 4 C overnight, followed by washing with PBS for 5 min for 3 times and incubation with secondary antibodies including donkey anti-goat Alexa 647 (1:300, A32849, Invitrogen), donkey anti-sheep Alexa 647 (1:300, A21448, Invitrogen), and donkey anti-rabbit Alexa 488 at room temperature for 30 min. For immunostaining of PDPN in human liver slides, we amplified fluorescence signal using the avidin-biotin amplification method (SP-2001, Vector Laboratories, Newark, CA).

Techniques: Imaging, Staining, Ligation

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver.

doi: 10.1152/ajpgi.00139.2023

Figure Lengend Snippet: Figure 5. A workflow for quantification of lymphatic vessel parameters in 3-D volume images. Cleared liver tissue was stained with LYVE1 (red) for lym- phatic vessels (LVs) and reconstructed three-dimensional (3-D) images of the portal tract in the mouse liver (EC-GFP reporter mouse) were created. The “Surface” rendering tool in Imaris software were used to determine a total volume of LYVE1-positive LVs (A–C), whereas the “Filament Tracer” rendering tool was used for quantification of the diameter and branch number of LVs (D–F). A:LYVE1-positive LVs were captured based on fluorescence intensities. B: thresholds were manually adjusted to determine LVs. C:Rendered LYVE1-positive LVs were visually checked to confirm proper vascular connections. LVs with improper connections and/or speckled background structure were removed manually. D: LYVE1-positive LVs were captured based on fluores- cence intensities using automatic filament and path detection in the “Filament Tracer” rendering tool. E: thresholds were manually adjusted to determine LVs. F: LVs with disconnected and speckled background structures were removed manually. All outputs of LV parameters were exported to Excel files for statistical analysis. EC-GFP, Endothelial-specific green fluorescent protein mice; LV, lymphatic vessel.

Article Snippet: The liver slides were washed with PBS and incubated in blocking buffer (PBS containing 5% donkey serum and 0.3% triton X-100) at room temperature for 1 h. After blocking was completed, the liver slides were incubated with goat anti-mouse LYVE1 (1:200, AF2125, R&D Systems, Minneapolis, MN) (17), sheep anti-rat LYVE1 (1:100, AF7939, R&D Systems) (18), and rabbit anti-COL-I (1:100, ab292, Abcam) (19) primary antibodies at 4 C overnight, followed by washing with PBS for 5 min for 3 times and incubation with secondary antibodies including donkey anti-goat Alexa 647 (1:300, A32849, Invitrogen), donkey anti-sheep Alexa 647 (1:300, A21448, Invitrogen), and donkey anti-rabbit Alexa 488 at room temperature for 30 min. For immunostaining of PDPN in human liver slides, we amplified fluorescence signal using the avidin-biotin amplification method (SP-2001, Vector Laboratories, Newark, CA).

Techniques: Staining, Software

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver.

doi: 10.1152/ajpgi.00139.2023

Figure Lengend Snippet: Figure 6. Comparison of lymphatic vessel parameters between 3-D volume-rendering images and conventional two-dimensional (2-D) images in normal and biliary fibrotic livers. A, left: reconstructed three-dimensional (3-D) image of the portal tract in the liver from a sham-operated mouse (EC-GFP re- porter mouse). Imaging condition: z-step size ¼ 0.4 μm step, image volume [x] [y] [z] ¼ 777 μm 777 μm 215 μm. Liver tissue was cleared and stained with LYVE1 (red, lymphatic vessel: LV) and aSMA (white, portal vein: PV). Right: 3-D volume-rendering image of lymphatic vessels and portal vein using Imaris software. Scale bars: 100 μm. B, left: reconstructed 3-D image of the portal tract in the liver from a 4-wk BDL mouse (EC-GFP reporter mouse). Imaging condition: z-step size ¼ 0.4 μm, image volume [x] [y] [z] ¼ 793 μm 894 μm 190 μm. Right: 3-D volume-rendering image of lym- phatic vessels and portal vein using Imaris software. Scale bars: 100 μm. C: quantification of lymphatic vessel (LV) parameters (diameter, branch number, and total volume) from 3-D volume-rendering images in sham (n ¼ 4) and 4-wk BDL (n ¼ 3) mouse livers. PT, portal tract; FC, fold change. D: conventional 2-D images of liver tissues (frozen section, 7-lm thickness) from sham and 4-wk BDL mice (EC-GFP reporter mice). Red: LYVE1-positive lymphatic vessel. Scale bars: 100 μm. E: quantification of LV parameters (diameter, number, and total area) from conventional 2-D images in sham (n ¼ 6) and 4-wk BDL (n ¼ 6) mouse livers. BDL, bile duct ligation; EC-GFP, Endothelial-specific green fluorescent protein mice; LV, lymphatic vessel.

Article Snippet: The liver slides were washed with PBS and incubated in blocking buffer (PBS containing 5% donkey serum and 0.3% triton X-100) at room temperature for 1 h. After blocking was completed, the liver slides were incubated with goat anti-mouse LYVE1 (1:200, AF2125, R&D Systems, Minneapolis, MN) (17), sheep anti-rat LYVE1 (1:100, AF7939, R&D Systems) (18), and rabbit anti-COL-I (1:100, ab292, Abcam) (19) primary antibodies at 4 C overnight, followed by washing with PBS for 5 min for 3 times and incubation with secondary antibodies including donkey anti-goat Alexa 647 (1:300, A32849, Invitrogen), donkey anti-sheep Alexa 647 (1:300, A21448, Invitrogen), and donkey anti-rabbit Alexa 488 at room temperature for 30 min. For immunostaining of PDPN in human liver slides, we amplified fluorescence signal using the avidin-biotin amplification method (SP-2001, Vector Laboratories, Newark, CA).

Techniques: Comparison, Imaging, Staining, Software, Ligation