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: bioRxiv

Article Title: Dual targeting of astrocytic and endothelial GLUT1 enables functional rescue in GLUT1 deficiency syndrome

doi: 10.64898/2026.03.04.709430

Figure Lengend Snippet: (A) Representative sagittal brain sections from wild-type mice immunostained for Glut1 (green) and counterstained with DAPI (cyan). Negative control sections were processed in parallel without the primary anti-Glut1 antibody. Scale bar, 5 mm.(B) High-magnification images of the thalamic ventral posteromedial nucleus (VPM) from 2-month-old Aldh1l1-CreERT2; Rosa26^LSL-tdTomato (flex tdTomato) mice. Endothelial cells were labeled with tomato lectin (green), astrocytes with tdTomato (magenta), Glut1 immunoreactivity is shown in grayscale, and nuclei are counterstained with DAPI (cyan). Yellow arrowheads indicate Glut1 signal associated with lectin-positive vessels, and yellow arrows indicate Glut1 signal in tdTomato-positive astrocyte somata. Scale bar, 50 µm. (C) Representative images from normal human hippocampus stained for GLUT1 (green) and the astrocytic marker GLT1 (magenta), with merged images and DAPI (cyan). White arrowheads indicate vascular GLUT1 staining. Region 1 and Region 2 correspond to the stratum oriens (or alveus) and the pyramidal cell layer, respectively. Negative control sections were processed without primary antibodies. Scale bar, 50 µm. Glut1/GLUT1 immunostaining was visualized using tyramide signal amplification (TSA).

Article Snippet: Tomato lectin (1:1000; Vector Laboratories, FL-1171) was used for vascular endothelial staining.

Techniques: Negative Control, Labeling, Staining, Marker, Immunostaining, Amplification

Journal: bioRxiv

Article Title: Dual targeting of astrocytic and endothelial GLUT1 enables functional rescue in GLUT1 deficiency syndrome

doi: 10.64898/2026.03.04.709430

Figure Lengend Snippet: (A) Reporter constructs containing Region d, with or without an upstream CMV enhancer element, were packaged into AAV-V1 capsids and administered to wild-type mice by retro-orbital injection. (B) Representative confocal images of cortical sections stained with lectin (vascular endothelial cells) and SOX9 (astrocytes). Top, AAV-V1 carrying Region d alone drove mCherry expression predominantly in vascular endothelial cells, with detectable expression in astrocytes. Bottom, addition of a CMV enhancer upstream of Region d increased reporter signal without an obvious change in cell-type distribution, resulting in a pattern that more closely recapitulates endogenous SLC2A1 expression. Yellow arrowheads indicate representative vascular endothelial cells, and yellow arrows indicate astrocytes. Scale bar, 100 µm.

Article Snippet: Tomato lectin (1:1000; Vector Laboratories, FL-1171) was used for vascular endothelial staining.

Techniques: Construct, Injection, Staining, Expressing

Journal: Frontiers in Neuroanatomy

Article Title: Search for marker proteins to assess blood–brain barrier development

doi: 10.3389/fnana.2026.1717532

Figure Lengend Snippet: BBB maturation stages in the postnatal cerebral cortex based on glial configuration. (A) 3D images of AQP4, Iba1, lectin, and DAPI staining in the rat cerebral cortex at P1–30 are shown. Scale bar indicates 30 μm. AQP4 gradually accumulated in the blood vessels and localized to the blood vessels after P15. The number of Iba1+ microglia significantly increased after P10 and peaked at P15, changing their shape from ameboid to ramified. (B) Temporal changes in biotin permeability are corrected to a maximum value of 100%. Typical images of P4 and P15 rat brain cortical regions stained with streptavidin are shown. A solid line was drawn perpendicular to the blood vessel to quantify the fluorescence, which is indicated by the dashed line, and the red fluorescence value was measured, as shown in the upper right graph. The traces show the average of 70–76 transverse vessel lines for the respective postnatal ages. The maximum fluorescence value was set at 100, and the sum of the fluorescence values 20 μm to the left and to the right was calculated. The peak became sharper as the age increased. Astrocyte and microglial coverage rates were corrected to a P30 value of 100%. BBB formation and maturation in rats are classified into three phases based on biotin leakage and astrocyte and microglial contact with blood vessels . The phases are as follows: the “immature stage” when biotin permeability is still high; the “organic formation stage” when glial structures around blood vessels are formed; and the “completion stage” when the structures around blood vessels become stable and the morphology of microglia changes to a ramified type. Abbreviations: AQP4: aquaporin 4; BBB: blood–brain barrier; DAPI: 4′,6-diamidino-2-phenylindole; Iba1: ionized calcium binding adapter protein 1; P: postnatal day.

Article Snippet: After rinsing, the sections were stained with lectin (1:200; DL1177; Vector Labs, Newark, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; 342-07431, Dojindo, Kumamoto, Japan).

Techniques: Staining, Permeability, Fluorescence, Binding Assay

Journal: Frontiers in Neuroanatomy

Article Title: Search for marker proteins to assess blood–brain barrier development

doi: 10.3389/fnana.2026.1717532

Figure Lengend Snippet: Expression of blood vessel developmental markers in the postnatal cerebral cortex. (A) Images of CD31, CD34, CD146, agrin, and Tie2 co-stained with lectin and DAPI in the rat cerebral cortex at P1–30 are shown. Scale bar indicates 50 μm. CD31 and Tie2 signals gradually increased in blood vessels, whereas CD34, CD146, and agrin signals gradually decreased. (B) The graphs show temporal changes in the percentage of marker+ area relative to the total vessel area. The colocalized signals between the markers and blood vessels were calculated, and the areas of the colocalized signals were normalized to the total blood vessel area in each image. Approximately 9–15 Images were obtained from the cerebral cortical regions of three rat pups of each age with a Nikon A1R-A1 confocal microscope. Data are shown as averaged value ± SEM. Data were analyzed using an ANOVA followed by a Tukey’s multiple range test. * p < 0.05, ** p < 0.01. vs. P1 value, while # p < 0.05, ## p < 0.01 vs. P10 value. (C) Graph summarizing results for B and B. Changes in biotin permeability, CD34, CD146, and agrin were corrected to a P1 value of 100%, and the astrocyte coverage rate, microglial coverage rate, CD31, and Tie2 were corrected to a P30 value of 100%. The grey zone represents the organic formation term (P4P15). Abbreviations: CD31, cluster of differentiation 31 or platelet endothelial cell adhesion molecule 1; CD146, melanoma cell adhesion molecule; DAPI: 4′,6-diamidino-2-phenylindole; P, postnatal day; Tie2, TEK receptor tyrosine kinase; ANOVA, analysis of variance.

Article Snippet: After rinsing, the sections were stained with lectin (1:200; DL1177; Vector Labs, Newark, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; 342-07431, Dojindo, Kumamoto, Japan).

Techniques: Expressing, Staining, Marker, Microscopy, Permeability

Journal: Frontiers in Neuroanatomy

Article Title: Search for marker proteins to assess blood–brain barrier development

doi: 10.3389/fnana.2026.1717532

Figure Lengend Snippet: Analysis of vascular TJ protein expression in the postnatal cerebral cortex. (A) Images of claudin-5, occludin, and ZO-1 co-stained with lectin and DAPI in the rat cerebral cortex at P1–30. Scale bar indicates 100 μm. Claudin-5 and occludin signals increased in blood vessels during development, whereas ZO-1 signals remained unchanged. (B) The graphs show changes in the percentage of marker+ area of the total vessel area at P4, P15, and P30. The colocalized signals between the markers and blood vessels were calculated, and the areas of the colocalized signals were normalized to the total blood vessel area in each image. Approximately 9–15 images were obtained from the cerebral cortical regions of three rat pups from each age group using a Nikon A1R-A1 confocal microscope. Data are shown as averaged value ± SEM. Data were analyzed using an ANOVA followed by a Tukey’s multiple range test. * p < 0.05, ** p < 0.01 vs. P4 value, and # p < 0.05 vs. P15 value. DAPI: 4′,6-diamidino-2-phenylindole; P, postnatal day; TJ, tight junction; ZO-1: zonula occludens-1; ANOVA, analysis of variance.

Article Snippet: After rinsing, the sections were stained with lectin (1:200; DL1177; Vector Labs, Newark, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; 342-07431, Dojindo, Kumamoto, Japan).

Techniques: Expressing, Staining, Marker, Microscopy

Journal: Frontiers in Neuroanatomy

Article Title: Search for marker proteins to assess blood–brain barrier development

doi: 10.3389/fnana.2026.1717532

Figure Lengend Snippet: Analysis of vascular expression of transporters and receptor proteins in the postnatal cerebral cortex. (A) Images of P-gp, BCRP, Glut1, and TfR co-stained with lectin and DAPI in the rat cerebral cortex at P1–30. Scale bar indicates 100 μm. The arrowhead shows the co-localization areas of the marker and lectin. P-gp and BCRP signals gradually increased in the blood vessels during development, whereas Glut1 and TfR signals were high in the blood vessels at P1 and remained unchanged. (B) The graphs show changes in the percentage of marker+ area of the total vessel area at P4, P15, and P30. The colocalized signals between the markers and blood vessels were calculated, and the areas of the colocalized signals were normalized to the total blood vessel area in each image. Approximately 9–15 images were obtained from the cerebral cortical regions of three rat pups from each age group using a Nikon A1R-A1 confocal microscope. Data are shown as averaged value ± SEM. Data were analyzed using an ANOVA followed by a Tukey’s multiple range test. * p < 0.05, ** p < 0.01 vs. P4 value. Abbreviations: BCRP: breast cancer resistance protein; DAPI: 4′,6-diamidino-2-phenylindole; Glut1: glucose transporter type 1; P: postnatal day; P-gp: P-glycoprotein; TfR: transferrin receptor; ANOVA, analysis of variance.

Article Snippet: After rinsing, the sections were stained with lectin (1:200; DL1177; Vector Labs, Newark, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; 342-07431, Dojindo, Kumamoto, Japan).

Techniques: Expressing, Staining, Marker, Microscopy

Journal: Science Advances

Article Title: A conserved glycan motif induces broadly reactive functional antibodies against the zoonotic pathogen Streptococcus suis

doi: 10.1126/sciadv.adz1854

Figure Lengend Snippet: ( A ) Binding of plant lectins to surface structures of capsule-deficient S. suis strains (ΔCPS). SBA binds to N -acetylgalactosamine (GalNAc) and, to a lesser extent, galactose (Gal); RCA 120 binds to both Gal and GalNAc; sWGA has a special affinity to N -acetylglucosamine (GlcNAc). Data from biological triplicates are presented as mean values ± SD. ( B ) Glycosyl composition analysis by GC-MS of TMS (trimethylsilyl) derivatives of methyl glycosides of S. suis RPS from S10 and 861160 released by mild acid hydrolysis after chemical N -acetylation. ( C ) Plant lectin binding to isolated RPS from S. suis S10 and 861160. Data show technical triplicates (mean values ± SD) and are representative for two independent experiments. ( D and E ) Presence and absence of the most abundant glycosyl linkage residues (D) and phosphate (E) of S. suis RPS from S10 and 861160. Glycosyl linkage residues were analyzed by GC-MS of partially methylated alditol acetate derivatives. Phosphate was determined by malachite green assay following hydrolysis with hydrochloric acid and digestion with alkaline phosphatase. Original data is in tables S2 and S3.

Article Snippet: Subsequently, 12.5 μl of bacterial suspension was incubated in a 96-well V-bottom plate with the same volume of pig serum or fluorescein-labeled lectin (Vector Laboratories, FLK-2100 and FL-1021S-5).

Techniques: Binding Assay, Gas Chromatography-Mass Spectrometry, Isolation, Methylation, Malachite Green Assay