Journal: iScience

Article Title: Comparative analysis of clearing methods for 3D imaging of the vasculature in mineralized mouse tissues

doi: 10.1016/j.isci.2026.115464

Figure Lengend Snippet: Overview of the experimental workflow (A–D) Animal is anesthetized (A), followed by retro-orbital lectin injection (B). Animals then undergo transcardiac perfusion (C), followed by leg dissection and skin removal (D). (E–H) (E) Leg samples undergo fixation, decalcification, delipidation methods, and RI matching (tissue clearing) to render them transparent, as shown in (F). Cleared leg samples were imaged on a light-sheet fluorescence microscope (G) to view the vasculature, as shown in (H).

Article Snippet: After the depth of anesthesia was confirmed by absence of toe pinch reflex, mice were retro-orbitally injected with 50 μL Lycopersicon esculentum (tomato) lectin 649 nm (Vector Laboratories, USA DL-1178-1) or 50 uL of 2% Evans blue dye (Sigma-Aldrich E2129) in sterile saline solution (Sigma-Aldrich S8776) into the retro-bulbar sinus vein using a 31-gauge needle.

Techniques: Injection, Dissection, Fluorescence, Microscopy

Journal: iScience

Article Title: Comparative analysis of clearing methods for 3D imaging of the vasculature in mineralized mouse tissues

doi: 10.1016/j.isci.2026.115464

Figure Lengend Snippet: Comparison of clearing methods on mouse hindlimb vascular visualization (A) Schematic diagram of the knee region indicating imaging orientation and planes of depth of view. (B–G) Light microscopy images of mouse hindlimbs cleared using either iDISCO + , vDISCO, fDISCO, EZ Clear, Binaree, or CLARITY. (H–M) Sagittal view of light-sheet fluorescent microscope (LSFM) images of mouse hindlimbs following perfusion with lectin-649 nm and processing with the indicated tissue clearing protocols (far left column). Yellow dashed box indicates the knee region. (N–S) Magnified view of the knee region corresponding to the samples shown in (H–M). (T–Y) Images showing the depth of view of the knee region (the yellow axis for the Z plane is indicated in each panel on the far left of the image). (Z–E′) Optical sections along the z axis of the knee region at increasing depths (from 1 to 3 mm) highlight the retention of crisp signal in the vessels within the iDISCO+ and EZ Clear processed samples. n = 5 samples per group; t test, p ≤ 0.05. Scale bars, 500 μm. See also , , , , , , and .

Article Snippet: After the depth of anesthesia was confirmed by absence of toe pinch reflex, mice were retro-orbitally injected with 50 μL Lycopersicon esculentum (tomato) lectin 649 nm (Vector Laboratories, USA DL-1178-1) or 50 uL of 2% Evans blue dye (Sigma-Aldrich E2129) in sterile saline solution (Sigma-Aldrich S8776) into the retro-bulbar sinus vein using a 31-gauge needle.

Techniques: Comparison, Imaging, Light Microscopy, Microscopy

Journal: iScience

Article Title: Comparative analysis of clearing methods for 3D imaging of the vasculature in mineralized mouse tissues

doi: 10.1016/j.isci.2026.115464

Figure Lengend Snippet: Evaluation of decalcification duration for achieving optimal clearing and vascular visualization in aged mouse hindlimbs (A) A sagittal maximum intensity projection following LSFM imaging of a mouse hindlimb perfused with lectin-649 nm and cleared using iDISCO + with 2 days of decalcification in 10% EDTA. The yellow dashed area is magnified in (B) and represents the knee region, with the outline of the femur and tibia noted. (C) A depth-of-view image of the sample in (A) (note the z axis, in yellow, at the far left) showing how fluorescence signal diminishes at greater depths. (D) A similarly perfused mouse hindlimb processed for iDISCO + clearing after 5 days of decalcification. (E) A magnified view of the knee region from (A) and (F) a depth-of-view image showing improved signal intensity overall, less signal from bone, and more intense signal at greater imaging depths along the z axis. (G) Schematic of the knee region showing imaging orientation and planes of optical sections shown in (H) and (I). (H and I) Comparison of optical sections of the knee along the z axis. (J) Quantification of the signal-to-background fluorescence ratio (SBR) (expressed as mean ± SEM) in the mouse hindlimb showing increased SBR in the 5-day decalcification samples compared to 2-day decalcification. n = 5 samples per group (6-month-old mice; both sexes); t test, ∗∗∗∗ p ≤ 0.0001. Scale bars, 500 μm. See also , and .

Article Snippet: After the depth of anesthesia was confirmed by absence of toe pinch reflex, mice were retro-orbitally injected with 50 μL Lycopersicon esculentum (tomato) lectin 649 nm (Vector Laboratories, USA DL-1178-1) or 50 uL of 2% Evans blue dye (Sigma-Aldrich E2129) in sterile saline solution (Sigma-Aldrich S8776) into the retro-bulbar sinus vein using a 31-gauge needle.

Techniques: Imaging, Fluorescence, Comparison

Journal: iScience

Article Title: Comparative analysis of clearing methods for 3D imaging of the vasculature in mineralized mouse tissues

doi: 10.1016/j.isci.2026.115464

Figure Lengend Snippet: Assessing the impact of imaging orientation between iDISCO + and EZ Clear in the mouse hindlimb (A and B) Schematics illustrate the different imaging orientations and planes of optical sections for (C–R). (C–F) Comparison of how an anterior or sagittal orientation of the sample relative to the microscope objective impacts fluorescence signal intensity and depth within the vasculature of the adult murine hindlimb following perfusion with lectin-649 and either EZ Clear or iDISCO + tissue clearing. (G–J) Optical sections of both views, with the femur and tibia indicated. (K–N) Depth-of-view images and (O–R) optical sections along the z axis of the knee region. Scale bars, 500 μm. SLGV, superior lateral geniculate vessel; SMGV, superior medial geniculate vessel; IMGV, inferior medial geniculate vessel; ILGV, inferior lateral geniculate vessel. n = 5 samples per group; t test, p ≤ 0.05. Scale bars, 500 μm. See also , , , , and .

Article Snippet: After the depth of anesthesia was confirmed by absence of toe pinch reflex, mice were retro-orbitally injected with 50 μL Lycopersicon esculentum (tomato) lectin 649 nm (Vector Laboratories, USA DL-1178-1) or 50 uL of 2% Evans blue dye (Sigma-Aldrich E2129) in sterile saline solution (Sigma-Aldrich S8776) into the retro-bulbar sinus vein using a 31-gauge needle.

Techniques: Imaging, Comparison, Microscopy, Fluorescence

Journal: iScience

Article Title: Comparative analysis of clearing methods for 3D imaging of the vasculature in mineralized mouse tissues

doi: 10.1016/j.isci.2026.115464

Figure Lengend Snippet: Comparison of the mouse hindlimb vascular network visualized by micro-CT or by iDISCO + clearing and light-sheet imaging (A and B) Anterior view of representative micro-CT images of the mouse hindlimb following perfusion with Vascupaint contrast agent and an LSFM image of a mouse hindlimb perfused with lectin-649 and cleared using iDISCO + . Bone in the micro-CT images is pseudocolored white, while vessels in both the micro-CT and light-sheet panels are color coded based on vessel diameter (the keys corresponding to vessel diameter are to the right of [E and F]). (C and D) Medial and (E and F) lateral views of the same samples. (G) Quantification of the frequency of different diameter vessels in micro-CT and LSFM-imaged samples, with error bars showing mean ± SEM. (H) Quantification of the difference in vessel volume relative to the sample volume (calculated as vessel volume ratio (%) = V e s s e l v o l u m e S a m p l e v o l u m e × 100%) between micro-CT and LSFM-imaged samples, with error bars showing mean ± SEM. F, femur; Fi, fibula; P, patella; T, tibia; IMGA, inferior medial geniculate artery; ILGA, inferior lateral geniculate artery; PA, popliteal artery; SMGA, superior medial genicular artery; SLGA, superior lateral genicular artery). n = 5 samples per group (2 month-old mice); t test, ∗∗∗∗ p ≤ 0.0001. Scale bars, 500 μm. See also .

Article Snippet: After the depth of anesthesia was confirmed by absence of toe pinch reflex, mice were retro-orbitally injected with 50 μL Lycopersicon esculentum (tomato) lectin 649 nm (Vector Laboratories, USA DL-1178-1) or 50 uL of 2% Evans blue dye (Sigma-Aldrich E2129) in sterile saline solution (Sigma-Aldrich S8776) into the retro-bulbar sinus vein using a 31-gauge needle.

Techniques: Comparison, Micro-CT, Imaging

Journal: Journal of leukocyte biology

Article Title: Activated NK cells kill hepatic stellate cells via p38/PI3K signaling in a TRAIL-involved degranulation manner.

doi: 10.1002/JLB.2A0118-031RR

Figure Lengend Snippet: FIGURE 5 IL-18/poly I:C-primed NK cells kill hepatic stellate cells in a TRAIL- involved degranulation manner. IL-18 and poly I:C-primed NK cells were treated by anti-TRAIL or isotype antibody before coculture. HSC death was measured (A), and NK cell degranulation was evaluated by CD107a expression (C). Soluble TRAIL was added to cell coculture, the cell death of HSCs (B) and CD107a expression of NK cells (D) was shown. Results were shown as mean ± SEM of 6 independent experiments performed with 6 different donor NK cells. *P < 0.05, **P < 0.01, ***P < 0.001, paired t-test. The kinetics of the activated NK cell-mediated LX2 cell apoptosis were observed by using live-cell imaging (E).

Article Snippet: Soluble TRAIL (R&D System; Catalog number: 375-TL-010) was added to HSCs half hour earlier than NK cells in coculture.

Techniques: Expressing, Live Cell Imaging

Journal: Journal of leukocyte biology

Article Title: Activated NK cells kill hepatic stellate cells via p38/PI3K signaling in a TRAIL-involved degranulation manner.

doi: 10.1002/JLB.2A0118-031RR

Figure Lengend Snippet: FIGURE 6 IL-18/poly I:C-primed hepatic NK cells kill HSCs in a TRAIL-involved degranulation manner. Liver NK cells were purified from speci- men, then primed by IL-18 and/or poly I:C before cocultured with primary HSCs as above. CD107a expression of liver NK cells (A) and PI/AnnexinV level of primary HSCs (B) were detected. Anti-TRAIL or isotype antibody were used to treat IL-18/poly I:C-primed liver NK cells before cocultured with primary HSCs, then CD107a expression of liver NK cells (C) and the cell death of primary HSCs (D) was measured. Results were shown as mean ± SEM of 5 independent experiments performed with 5 different donor NK cells. *P < 0.05, **P < 0.01, paired t-test.

Article Snippet: Soluble TRAIL (R&D System; Catalog number: 375-TL-010) was added to HSCs half hour earlier than NK cells in coculture.

Techniques: Purification, Expressing

Journal: bioRxiv

Article Title: Metabotropic NMDA Receptor Signaling Contributes to Sex Differences in Synaptic Plasticity and Episodic Memory

doi: 10.1101/2024.01.26.577478

Figure Lengend Snippet: a-e) Electrode placements as in . ( a ) MK801 (30µM) blocked TBS-induced LTP in female rats (vehicle, veh N=5, MK801 N=4). ( b ) Phalloidin labeling in CA1 of slices that received control, low frequency SC stimulation (con) or 10 burst TBS in the presence of vehicle, MK801, or Ro25 (3µM). ( c ) TBS increased phalloidin labeled spine F-actin levels in the presence of vehicle, MK801, or Ro25 (vs. controls) but this increase was blocked by NMDAR antagonist APV (F 4,62 =22.88, p<0.0001; N=5-33, values normalized to con mean). ( d ) Ro25 did not disrupt TBS-induced LTP in female slices (veh N=5, Ro25 N=6). Traces from before (solid) and 60 min after (dashed) TBS. ( e ) In vehicle-slices, TBS increased numbers of densely phalloidin labeled puncta in both sexes. This effect was blocked by ERα antagonist MPP (3µM) in females (F 2,29 =16.02, p<0.0001; N=6-17) but not in males (F 2,21 =20.28, p<0.0001; N=6-12). ( f ) Deconvolved images of NMDAR subunit and PSD95 immunolabeling in CA1; arrows indicate double-labeled profiles. In females, the % PSD95 + synapses with dense GluN1-immunoreactivity (ir) was greater than in males (p=0.0171) whereas levels of GluN2A- and GluN2B-ir were comparable (N=17-20/group). The percent PSD95 + synapses with dense pGluN2B Y1472-ir was lower in females than males (p=0.0041; N=17-20/group). ( g ) Mice received vehicle, Ro25 or MPP before odor exposure in the 4-corner episodic ‘where’ paradigm. ( h,i ) Vehicle-treated males and females (2 cohorts) discriminated the moved cues in the episodic ‘where’ task; ( h ) Ro25 disrupted this effect in males (veh N=5, Ro25 N=4) and had no effect on female performance (F 1,15 =19.62, p=0.0005; female N=5/group). ( i ) In contrast, MPP blocked this ‘where’ acquisition in females but did not attenuate performance in males (F 1,24 =8.001, p=0.0093; N=7/group). Scale bar: ( a,d ) 1mV, 10ms; ( b,e ) 5μm; ( f ) 2μm. Statistics: two-tailed unpaired t-test ( a,d ), ( f ) two-tailed unpaired t-test Welch’s correction, ( c,e ) one-way ANOVA with Tukey post-hoc, ( h,i ) 2-way ANOVA with post-hoc Tukey. Asterisks inside bars denote comparison to controls; n.s. = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.00001. Mean ± s.e.m values shown. Table S1 contains detailed statistics.

Article Snippet: MPP (3μM; Tocris, 1991) and Ro25-6981 (3μM; Hello Bio, HB0554) were dissolved with DMSO (≤0.01%).

Techniques: Labeling, Control, Immunolabeling, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Metabotropic NMDA Receptor Signaling Contributes to Sex Differences in Synaptic Plasticity and Episodic Memory

doi: 10.1101/2024.01.26.577478

Figure Lengend Snippet: ( a ) Schematic detailing the protocols (retention data is presented in for Ro25 and for MPP). ( b-e ) Graphs show that the sampling times (b,d), and the velocity and distance traveled (c,e), for all drug-treated groups were not lower than their respective vehicle-treated groups; the Ro25 drug was found to increase sampling times in males during the test phase (b) but other measures were not affected (p-values at top of columns are for vehicle vs. drug post-hoc comparisons for each measure; n.s., not significant, p>0.05). In addition, across all measures there was no statistical difference for effect of drug between sexes during either training or testing. For both training and test phases, data were analyzed by 2-way ANOVA (sex and drug) followed by Tukey post hoc comparisons; See Table S1 for detailed statistics. Mean and s.e.m. shown.

Article Snippet: MPP (3μM; Tocris, 1991) and Ro25-6981 (3μM; Hello Bio, HB0554) were dissolved with DMSO (≤0.01%).

Techniques: Sampling

Journal: PLoS ONE

Article Title: TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

doi: 10.1371/journal.pone.0105627

Figure Lengend Snippet: After 12 days, beads were removed and the cells set up in new media. Cytokines were added in the following concentrations IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL, TL1A Ab: 1 µg/mL, CsA 1 µg/mL. Pictures using the light microscope were taken at 25× magnification.

Article Snippet: Cytokines, blocking TL1A antibody (TL1AAb) and Cyclosporine A (CsA) were added in the following concentrations: IL-12 (RnD Systems, Cat# 219-IL): 4 ng/mL, IL-15 (Peprotech, Cat# 200-15): 10 ng/mL, IL-18 (MBL, Cat# B003-5): 40 ng/mL, TL1A (RnD Systems, Cat# 1319-TL): 100 ng/mL, TL1AAb (RnD Systems, Cat# MAB7441): 1 μg/mL, CsA (Sigma-Aldrich, Cat# C1832):1 μg/mL.

Techniques: Light Microscopy

Journal: PLoS ONE

Article Title: TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

doi: 10.1371/journal.pone.0105627

Figure Lengend Snippet: Purified PBLs were stimulated for 12 days with CD3/CD28 beads. After 12 days, beads were removed and the cells set up in new media. Cytokines were added in the following concentrations IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL, TL1A Ab: 1 µg/mL. Surface expression of co-receptors and activation markers CD25, CD134, CD154 and LFA-1 after 72 h. Upper panels: Gating strategy for lymphocytes. Lower panels: Co-staining for CD4 and CD134, CD154, CD25 of LFA-1, % positive cells and MFI for upper right quadrant are shown. Data are representative of results obtained with cells from three different donors.

Article Snippet: Cytokines, blocking TL1A antibody (TL1AAb) and Cyclosporine A (CsA) were added in the following concentrations: IL-12 (RnD Systems, Cat# 219-IL): 4 ng/mL, IL-15 (Peprotech, Cat# 200-15): 10 ng/mL, IL-18 (MBL, Cat# B003-5): 40 ng/mL, TL1A (RnD Systems, Cat# 1319-TL): 100 ng/mL, TL1AAb (RnD Systems, Cat# MAB7441): 1 μg/mL, CsA (Sigma-Aldrich, Cat# C1832):1 μg/mL.

Techniques: Purification, Expressing, Activation Assay, Staining

Journal: PLoS ONE

Article Title: TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

doi: 10.1371/journal.pone.0105627

Figure Lengend Snippet: Purified PBLs were stimulated for 12 days with CD3/CD28 beads. After 12 days, beads were removed and the cells set up in new media. Cytokines/inhibitors were added in the following concentrations IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL, TL1A Ab: 1 µg/mL, CsA 1 µg/mL. Surface markers were measured at t = 6, 24, 72, 96, 168 and 240 h. Cells/µL was multiplied by MFI (cells/µL*MFI) to take both expression levels and cell growth into account. Data are representative of results obtained with cells from three different donors.

Article Snippet: Cytokines, blocking TL1A antibody (TL1AAb) and Cyclosporine A (CsA) were added in the following concentrations: IL-12 (RnD Systems, Cat# 219-IL): 4 ng/mL, IL-15 (Peprotech, Cat# 200-15): 10 ng/mL, IL-18 (MBL, Cat# B003-5): 40 ng/mL, TL1A (RnD Systems, Cat# 1319-TL): 100 ng/mL, TL1AAb (RnD Systems, Cat# MAB7441): 1 μg/mL, CsA (Sigma-Aldrich, Cat# C1832):1 μg/mL.

Techniques: Purification, Expressing

Journal: PLoS ONE

Article Title: TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

doi: 10.1371/journal.pone.0105627

Figure Lengend Snippet: A. Purified PBLs were stimulated for 12 days with CD3/CD28 beads, after which cells were stimulated with combinations of cytokines. Cytokines/inhibitors were added in the following concentrations: IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL, TL1A Ab: 1 µg/mL, CsA: 1 µg/mL Expression of surface markers was measured by flow cytometry after 96 h. Cells/µL was multiplied by MFI (cells/µL*MFI) to take both expression levels and cell growth into account. B. Freshly purified PBLs were stimulated with Cytokines/inhibitors in the following concentrations: IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL, TL1A Ab: 1 µg/mL, CsA: 1 µg/mL. Expression of surface markers was measured by flow cytometry after 72 h. Please note that the Y-axis of the graphs differ in range. Data are representative of results obtained with cells from three different donors.

Article Snippet: Cytokines, blocking TL1A antibody (TL1AAb) and Cyclosporine A (CsA) were added in the following concentrations: IL-12 (RnD Systems, Cat# 219-IL): 4 ng/mL, IL-15 (Peprotech, Cat# 200-15): 10 ng/mL, IL-18 (MBL, Cat# B003-5): 40 ng/mL, TL1A (RnD Systems, Cat# 1319-TL): 100 ng/mL, TL1AAb (RnD Systems, Cat# MAB7441): 1 μg/mL, CsA (Sigma-Aldrich, Cat# C1832):1 μg/mL.

Techniques: Purification, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

doi: 10.1371/journal.pone.0105627

Figure Lengend Snippet: Purified PBLs were stimulated for 12 days with CD3/CD28 beads. After 12 days, beads were removed and the cells set up in new media. Cytokines/inhibitors were added in the following concentrations IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL, TL1A Ab: 1 µg/mL, CsA 1 µg/mL Supernatant was collected 96 h after stimulation and cytokines measured by ELISA or multiplex as described in Materials and Methods. Mean +/− SEM on two measurements is shown. The addition of TL1A significantly increased the expression of cytokines compared to all other cytokine combinations tested;α IL-22 (P<0.0002) and GM-CSF (P<0.00005), t-test. Data are representative of results obtained with cells from three different donors.

Article Snippet: Cytokines, blocking TL1A antibody (TL1AAb) and Cyclosporine A (CsA) were added in the following concentrations: IL-12 (RnD Systems, Cat# 219-IL): 4 ng/mL, IL-15 (Peprotech, Cat# 200-15): 10 ng/mL, IL-18 (MBL, Cat# B003-5): 40 ng/mL, TL1A (RnD Systems, Cat# 1319-TL): 100 ng/mL, TL1AAb (RnD Systems, Cat# MAB7441): 1 μg/mL, CsA (Sigma-Aldrich, Cat# C1832):1 μg/mL.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Expressing

Journal: PLoS ONE

Article Title: TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

doi: 10.1371/journal.pone.0105627

Figure Lengend Snippet: Purified PBLs were stimulated for 12 days with CD3/CD28 beads. After 12 days, beads were removed and the cells set up as PBLs or CD8 depleted PBLs in new media. A. Left panel: Gating strategy for lymphocytes. Middle panel: Single cells. Right panel: Verification of CD8 depletion by staining for CD4/CD3 (∼95% were CD3 + CD4 + ). B. IL-22 and GM-CSF production by PBLs and CD8 depleted cells from A. Supernatant was collected seven days after stimulation and cytokines measured by ELISA as described in Materials and Methods. Cytokines/inhibitors were added in the following concentrations IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL. Mean +/− SEM on two measurements is shown. Data are representative of results obtained with cells from two different donors.

Article Snippet: Cytokines, blocking TL1A antibody (TL1AAb) and Cyclosporine A (CsA) were added in the following concentrations: IL-12 (RnD Systems, Cat# 219-IL): 4 ng/mL, IL-15 (Peprotech, Cat# 200-15): 10 ng/mL, IL-18 (MBL, Cat# B003-5): 40 ng/mL, TL1A (RnD Systems, Cat# 1319-TL): 100 ng/mL, TL1AAb (RnD Systems, Cat# MAB7441): 1 μg/mL, CsA (Sigma-Aldrich, Cat# C1832):1 μg/mL.

Techniques: Purification, Staining, Enzyme-linked Immunosorbent Assay