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: Retrovirology

Article Title: Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-κB activity: a comparative study with the HTLV-1 Tax-1 protein

doi: 10.1186/1742-4690-9-102

Figure Lengend Snippet: The intracellular distribution of Tax - 2B depends on the level and cellular context for expression. HeLa or 293T cells were transfected with high level (pJFE) or low level (pcDNA) expression vectors for either Tax-1 or Tax-2B and analyzed by immunofluorescence staining and confocal microscopy with anti-Tax-1 IgG2a monoclonal antibody or anti-Tax-2B rabbit polyclonal antibody followed by anti-rabbit or anti-mouse IgG antibodies conjugated to Dylight 488. DIC, differential inference contrast.

Article Snippet: The secondary antibodies used for immunofluorescence staining were Dylight 488-conjugated goat anti-mouse IgG2a, Dylight 549- or 488-conjugated goat anti-rabbit IgG and Dylight 649-conjugated goat anti-mouse IgG1 from Jackson ImmunoResearch.

Techniques: Expressing, Transfection, Immunofluorescence, Staining, Confocal Microscopy

Journal: Retrovirology

Article Title: Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-κB activity: a comparative study with the HTLV-1 Tax-1 protein

doi: 10.1186/1742-4690-9-102

Figure Lengend Snippet: Ubiquitination and sumoylation deficiency alters the intracellular localization of both Tax-1 and Tax-2B. 293T cells were cotransfected with vectors expressing wild type or mutant Tax-1 or Tax-2B and the transcriptional coactivator p300-HA. The cells were fixed, stained by triple immunofluorescence staining and analyzed by laser scanning confocal microscopy. Staining of Tax-1 expressing cells was with anti-Tax-1 IgG2a monoclonal antibody, anti-HA rabbit polyclonal antibody for the detection of p300-HA and anti-RelA IgG1 monoclonal antibody. For cells expressing Tax-2B, anti-Tax-2B rabbit polyclonal antibody and anti-HA monoclonal IgG2a antibody for the detection of p300-HA and the anti-RelA IgG1 monoclonal antibody were used. The secondary antibodies were goat anti-mouse IgG2a conjugated to Dylight 488, goat anti-rabbit IgG conjugated to Dylight 549 and goat anti-mouse IgG1 conjugated to Dylight 649. The profiles of the intensity of the fluorescence staining along lines crossing the nuclei are shown in the right panels (NE: nuclear envelope). The percentages of ubiquitination and sumoylation obtained in Figure are indicated for each mutant (Ub/SUMO). White arrows point to cytoplasmic structures that contain Tax-1 and RelA at the boundary of the nucleus.

Article Snippet: The secondary antibodies used for immunofluorescence staining were Dylight 488-conjugated goat anti-mouse IgG2a, Dylight 549- or 488-conjugated goat anti-rabbit IgG and Dylight 649-conjugated goat anti-mouse IgG1 from Jackson ImmunoResearch.

Techniques: Ubiquitin Proteomics, Expressing, Mutagenesis, Staining, Immunofluorescence, Confocal Microscopy, Fluorescence

Journal: Retrovirology

Article Title: Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-κB activity: a comparative study with the HTLV-1 Tax-1 protein

doi: 10.1186/1742-4690-9-102

Figure Lengend Snippet: Fusion of ubiquitin or SUMO - 1 to the C - terminus of wild type or K1 - 10iR mutant modifies their intracellular localization and increases NF -κ B activity. ( A ) 293T cells were cotransfected with vectors expressing wild type or K1-10iR Tax-2B mutant, fused to ubiquitin (Tax-2-Ub and Tax-2 K1-10iR-Ub) or SUMO-1 (Tax-2-SUMO and Tax-2 K1-10iR-SUMO). The cells were fixed and stained by dual immunofluorescence staining with anti-Tax-2B rabbit polyclonal antibody and anti-RelA IgG1 monoclonal antibody. The secondary antibodies were goat anti-rabbit IgG conjugated to Dylight 549 and goat anti-mouse IgG1 conjugated to Dylight 649. The images were collected using a laser scanning confocal microscope. White arrows point to cytoplasmic structures at the boundary of the nucleus in which Tax-2-Ub and Tax-2 K1-10iR-Ub fusions colocalize with RelA. DIC, differential inference contrast. ( B ) 293T cells were cotransfected with 50 ng of vectors expressing wild type or K1-10iR mutant Tax-2B fused or not to ubiquitin or SUMO-1 and 250 ng of the NF-κB-Luc reporter construct. 50 ng of the phRG-TK Renilla Luciferase vector was added to normalize for transfection efficiency. Cells were lysed and the luciferase activity was measured in each extracts. The activity of each mutant fusion is expressed as percentages relatively to the equivalent wild type Tax-2-Ub or Tax-2-SUMO fusions. The values were normalized for equal quantity of Tax proteins in each extract. The values reported are the averages of three independent experiments.

Article Snippet: The secondary antibodies used for immunofluorescence staining were Dylight 488-conjugated goat anti-mouse IgG2a, Dylight 549- or 488-conjugated goat anti-rabbit IgG and Dylight 649-conjugated goat anti-mouse IgG1 from Jackson ImmunoResearch.

Techniques: Ubiquitin Proteomics, Mutagenesis, Activity Assay, Expressing, Staining, Immunofluorescence, Microscopy, Construct, Luciferase, Plasmid Preparation, Transfection

Journal: Biomaterials

Article Title: Protease-Degradable Microgels for Protein Delivery for Vascularization

doi: 10.1016/j.biomaterials.2016.10.044

Figure Lengend Snippet: Microgels functionalized with a fluorescent RGD molecule were injected into the subcutaneous space on the back of C57BL/6 mice. (a) Images of microgel fluorescence at day 0 and day 14. (b) Percent of signal remaining over time compared to day 0. Data was fit with a simply decay model. (c) Representative fluorescent images of skin explants perfused with lectin to label vasculature. Fluorescent images of microgels in skin explants (bottom left). Dotted white line indicates the area represented in the large image. (d) Number of lectin labeled vessels per field of view (n = 4 mice per group). Significance was determined using one-way ANOVA with Tukey post-test, ***p<0.005.

Article Snippet: At 14 day, following injection, functional vasculature was labeled by perfusing anesthetized mice with 1.0 mg/mL Dylight649-labeled tomato lectin (Vector Labs) via tail vein injection.

Techniques: Injection, Fluorescence, Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Distinct miRNA profiles in human amniotic tissue and its vesicular and non-vesicular secretome

doi: 10.3389/fcell.2025.1692501

Figure Lengend Snippet: Characterization of hAM-derived extracellular vesicles. (A) Particle size distribution by nanoparticle tracking analysis (NTA) showed that most particles were ≤200 nm; n = 3, mean ± SEM. (B) Median values (X50) of particle size range from 138 nm to 211 nm after 72 h of incubation (x̄ = 177 in RA and x̄ = 174 in P), n = 3. The majority of particles were ≤200 nm. X50 values are indicated ±SD. (C) Fluorescence-triggered flow cytometry analysis (FT-FC) showed that 20%–35% of the EVs were positive for CD81 (x̄ = 19.55 in RA, x̄ = 24.40 in P). The expression of CD9 was less pronounced n = 2. (D) Western Blot analysis showed that CD63 and CD81 were more abundant in EVs than tissue, n = 3. (E) TEM confirmed EV morphologies and CD81 positive particles (immuno-gold particles (black dots)) in EV preparations from RA and P. scale bar = 100 nm. Abbreviations: human amniotic membrane (hAM), extracellular vesicles (EV), placental amnion (P), reflected amnion (RA), flotillin-1 (Flot-1), lysosomal associated membrane protein 1 (LAMP1), heat shock protein 70 (HSC70), tumor susceptibility gene 101 (TSG101), cluster of differentiation (CD), transmission electron microscopy (TEM).

Article Snippet: The staining of the respective antigens was performed by subsequent addition of 4 μL of 1:100 predilute PE-labelled antibodies for CD9 (Miltenyi Biotech, 130-118-865), CD63 (Miltenyi Biotech, Germany, 130-118-077), CD81 (Miltenyi Biotech, Germany, 130-118-481), and isotype IgG1 (Miltenyi Biotech, Germany, 130-113-438).

Techniques: Derivative Assay, Incubation, Fluorescence, Flow Cytometry, Expressing, Western Blot, Membrane, Transmission Assay, Electron Microscopy