Journal: Nature Communications

Article Title: Periarteriolar spaces modulate cerebrospinal fluid transport into brain and demonstrate altered morphology in aging and Alzheimer’s disease

doi: 10.1038/s41467-022-31257-9

Figure Lengend Snippet: Routine ( a ), ultrastructural ( b , c ), and super-resolution fluorescent ERTR7 labeled ( d ) images of the brain surface demonstrate the reticulated nature of pial cells. On coronal section ( e ), the proximal middle cerebral artery is observed to be thickly ensheathed (open arrow), while a distal oblique arterial segment exhibits a thin sheath (solid arrow) and small arterial branches are unsheathed by ERTR7. Large subarachnoid arteries in ventral brain region are depicted on longitudinal ( e ) and cross ( e , f ) sections. Enlargement of the boxed area in f highlights ERTR7-positive tunica adventitia in apposition to the arterial tunica media ( g ); insets at upper right are representative of the boxed area and show punctate ERTR7 label, consistent with matted pial cell processes. Partial pial ensheathment is appreciated on cross section of a small artery ( h ), and enlargement of the boxed area ( i ). Diagram depicting brain regions used for quantification ( j ). Analysis of epipial coverage according to anatomical brain region ( k ) and vessel size ( l , m ); n = 145 vessels from 6 mice. a H&E; b transmission electron micrograph; c Immuno-EM for ERTR7; d – i green/FITC, ERTR7; red/CY3, SMA; blue, DAPI; scale bars = ( a – d ) 2 μm; c and d (insets) 200 nm; e , f 50 μm; g – i 10 μm. Primary (1 o ), secondary (2 o ), and tertiary (3 o ) processes are depicted within pial cells ( b , d ). Source data are provided as a Source Data file. TEM data are representative of 50 vessels from 3 young mice and immuno-EM data are representative of 6 sections from 2 young mice.

Article Snippet: Samples were permeabilized in 0.1% Triton X-100 with 5% goat serum (Sigma) for 2 h and then incubated for two days at 4 °C with a monoclonal rat ERTR7 antibody (NB100-64932, Novus Biologicals, Centennial CO, 1:1200).

Techniques: Labeling, Transmission Assay

Journal: Nature Communications

Article Title: Periarteriolar spaces modulate cerebrospinal fluid transport into brain and demonstrate altered morphology in aging and Alzheimer’s disease

doi: 10.1038/s41467-022-31257-9

Figure Lengend Snippet: Schematic ( a ) and routine sections ( b – d ) demonstrate the relationships of pial cells on leptomeningeal, i.e., subarachnoid space (SAS) arteries and penetrating arterioles. Medium-sized SAS arteries at the brain surface demonstrate attenuation and loosening of the epipia layer, creating an epipial space ( e ), whereas smaller SAS arteries and penetrating arterioles demonstrate further thinning and coalescence of pial layers with the vessel walls, with occasional envelopment of the arteriolar smooth muscle cell layer ( f , g ). Analysis of epipial sheath thickness, expressed as a percentage (%) of vessel area, is shown according to vessel size ( h ) and brain region ( i ); Pearson correlation coefficient; n = 86 vessels. Likewise, analysis of epipial fenestration, expressed as a percentage (%) of total vessels, is shown according to brain region ( j ) and vessel size ( k ); one-way ANOVA with Tukey’s multiple comparisons test; n = 122 vessels. A medium-sized artery in ventral mouse brain ( l , the same vessel shown in e ), is depicted at higher magnification and demonstrates the loosened epipial sleeve composed of interlinking pial cells that partially enclose the epipial space (asterisks). Features of the epipial space are highlighted by immunohistochemistry ( l , right-hand side), and are shown to advantage in ultrastructural images ( m ). Enlargements of boxed micrograph areas demonstrate the intra-adventitial space, in which scattered collagen fibrils are appreciated (arrows). Analysis of epipial space areas are shown relative to vessel size ( n ); Pearson correlation coefficient; n = 115 vessels from 6 mice. Analysis of epipial space areas are shown relative to brain region ( o ); one-way ANOVA with Tukey’s multiple comparisons test; n = 115 vessels from 6 mice. ( b – d , L left) H&E; ( e – g , L right) red/CY3, SMA; green/FITC, ERTR7; blue, DAPI; m transmission electron micrographs, with scale bars as indicated (asterisks represent the epipial space); scale bars = ( b ) 100 μm; c – e 20 μm; f , g 10 μm. Source data are provided as a Source Data file. TEM data are representative of 50 vessels from 3 mice.

Article Snippet: Samples were permeabilized in 0.1% Triton X-100 with 5% goat serum (Sigma) for 2 h and then incubated for two days at 4 °C with a monoclonal rat ERTR7 antibody (NB100-64932, Novus Biologicals, Centennial CO, 1:1200).

Techniques: Immunohistochemistry, Transmission Assay

Journal: Nature Communications

Article Title: Periarteriolar spaces modulate cerebrospinal fluid transport into brain and demonstrate altered morphology in aging and Alzheimer’s disease

doi: 10.1038/s41467-022-31257-9

Figure Lengend Snippet: As shown on volumetric lightsheet image of a tissue-cleared (CLARITY) specimen, the intimal pia and epipia adjoin at the brain surface and create a basket-like sieve around penetrating arterioles ( a ). The anatomy is further delineated on confocal Z-stack images of longitudinal and axial cortical mouse brain sections ( b ). Heterogeneous relationships of the intimal pia and epipia around penetrating arterioles result in a spectrum of periarteriolar space (PAS) anatomy, with three primary structures in healthy young mouse brains: Type A ( c ), Type B ( d ), or Type C ( e ); white arrows represent separate pial sheaths (i.e., the intimal pia and epipia), while arrowheads mark sites of pial coalescence with the tunica media. The variable anatomy are depicted in longitudinal images with enlargements of boxed areas ( c – e , left-hand side) and axial schematics ( c – e , right-hand side) that depict deeper levels, from left to right (labeled 1–4, respectively). Type A PAS is shown in relation to an unsheathed arteriole (i.e., an arteriole lacking epipial coverage at the site of brain penetration). The distribution of PAS types at the brain surface is summarized in ( f ). PAS types are further characterized according to brain region ( g ) and vessel size ( h and i ); one-way ANOVA with Tukey’s multiple comparisons test; n = 57 vessels from 6 mice. a – e Red/CY3, SMA; green/FITC, ERTR7; blue, DAPI or lectin; violet/CY5, aquaporin 4; scale bars = a , e 50 μm; b , c 10 μm; d 30 μm. Source data are provided as a Source Data file.

Article Snippet: Samples were permeabilized in 0.1% Triton X-100 with 5% goat serum (Sigma) for 2 h and then incubated for two days at 4 °C with a monoclonal rat ERTR7 antibody (NB100-64932, Novus Biologicals, Centennial CO, 1:1200).

Techniques: Labeling

Journal: Nature Communications

Article Title: Periarteriolar spaces modulate cerebrospinal fluid transport into brain and demonstrate altered morphology in aging and Alzheimer’s disease

doi: 10.1038/s41467-022-31257-9

Figure Lengend Snippet: Distribution of an immunofluorescent tracer (Texas Red conjugated to bovine serum albumin, TxRd; mw 66 kDa) was evaluated in mice sacrificed at 15 versus 30 min following slow intracisternal infusion ( a ), and revealed heterogeneous spatiotemporal deposition patterns as shown in right-hand images that represent the boxed areas. Enlargement of the small box at lower right panel in a illustrates tracer passage into a Type B PAS ( b ). Enlarged inset depicts pooling of tracer in PAS, creating a scalloped appearance around smooth muscle cells ( b , inset). Cross-sectional images from axial sections (labeled 1–3) of another Type B PAS is shown in the middle panel and the anatomic relationships are summarized in axial schematics on right-hand side. Triple labeling of a Type B penetrating arteriole (shown in longitudinal section) depicts TxRd tracer signal around the ERTR7-labeled pial cell elements ( c ). Volumetric lightsheet image of a tissue-cleared (CLARITY) specimen further demonstrates variable penetration of the tracer ( d ). Examples of tracer-positive PAS are shown ( e ) and distributions are quantified in different brain regions at 15 min ( f , h ) and 30 min ( g , i ) post-infusion; n = 57 vessels from 6 mice. j At 30 min post-infusion, tracer was mostly found around large diameter arteries; n = 141 vessels from 6 mice. Red/Texas Red, TxRd; green/FITC, ERTR7; white/CY5, SMA; blue, DAPI or lectin; scale bars = a 500 μm; b – e 10 μm. Source data are provided as a Source Data file.

Article Snippet: Samples were permeabilized in 0.1% Triton X-100 with 5% goat serum (Sigma) for 2 h and then incubated for two days at 4 °C with a monoclonal rat ERTR7 antibody (NB100-64932, Novus Biologicals, Centennial CO, 1:1200).

Techniques: Labeling

Journal: Nature Communications

Article Title: Periarteriolar spaces modulate cerebrospinal fluid transport into brain and demonstrate altered morphology in aging and Alzheimer’s disease

doi: 10.1038/s41467-022-31257-9

Figure Lengend Snippet: ERTR7-labeled coronal brain sections from young (2 months), old (13 months), and APP/PS1 (13 months) mice demonstrate pial coverage at the brain surface ( a ). Notice variable coverage, thickness, and depths of penetration. Analyses of percent (%) intimal pial/surface coverage ( b ), intimal pial/surface thickness (µm) ( c ), and depth of pial penetration (µm) according to brain region ( d ), and PAS type ( e ) are shown; two-way ANOVA with Tukey’s multiple comparisons test. The overall distribution of pial depths and areas (µm 2 ) of pial coverage surrounding penetrating arterioles are shown in plots f and g , respectively. Young: n = 141 vessels from 3 mice; Old: n = 139 vessels from 3 mice; APP/PS1: n = 141 vessels from 3 mice. Green/FITC, ERTR7; cyan/CY5, MeX04; blue, lectin; scale bars = a – c , left 500 μm; a – c , right 50 μm. Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Samples were permeabilized in 0.1% Triton X-100 with 5% goat serum (Sigma) for 2 h and then incubated for two days at 4 °C with a monoclonal rat ERTR7 antibody (NB100-64932, Novus Biologicals, Centennial CO, 1:1200).

Techniques: Labeling

Journal: Nature Communications

Article Title: Periarteriolar spaces modulate cerebrospinal fluid transport into brain and demonstrate altered morphology in aging and Alzheimer’s disease

doi: 10.1038/s41467-022-31257-9

Figure Lengend Snippet: In old (13 months) mice, pial distribution is irregular at the brain surface and around penetrating arterioles ( a ), resulting in PAS without any pial coverage, i.e., Type 0 ( b ), and PAS with thickened, superficial plaque-like coverage, i.e., Type D ( c ), in addition to usual type PAS (i.e., Type a – c ). The anatomy of Type 0 and Type D PAS are depicted in schematics shown in ( d ) and ( e ), respectively. PAS types observed in old and APP/PS1 mice are illustrated in ( f ) and ( g ). The overall distribution of PAS types at the cerebral cortical brain surface of old mice is summarized in ( h ). The overall distribution of PAS types at the cerebral cortical brain surface of APP/PS1 mice is summarized in ( i ). Note diminishment of type 0 and absence of type C PAS in APP/PS1 mice. Old: n = 139 vessels from 3 mice; APP/PS1: n = 141 vessels from 3 mice. Cyan/CY5, MeX04; blue, lectin; green/FITC, ERTR7. Scale bars = a 50 μm; b , c 10 μm; f , g 20 µm.

Article Snippet: Samples were permeabilized in 0.1% Triton X-100 with 5% goat serum (Sigma) for 2 h and then incubated for two days at 4 °C with a monoclonal rat ERTR7 antibody (NB100-64932, Novus Biologicals, Centennial CO, 1:1200).

Techniques:

Journal: Nature Communications

Article Title: Periarteriolar spaces modulate cerebrospinal fluid transport into brain and demonstrate altered morphology in aging and Alzheimer’s disease

doi: 10.1038/s41467-022-31257-9

Figure Lengend Snippet: Coronal images demonstrate TxRd tracer accumulation in brains of young, old and APP/PS1 mice ( a ). The distribution of vessel sizes in young, old and APP/PS1 mice are shown ( b ) along with the proportion of tracer-positive PAS ( c ) and tracer positivity according to PAS type ( d ). Distribution of tracer-positive PAS are further depicted in the three groups according to arterial diameter ( e ). The overall distribution of tracer mean pixel intensity (MPI) is shown ( f ) along with distribution of MPI according to PAS type ( g ); two-way ANOVA with Tukey’s multiple comparisons test. A penetrating arteriole from APP/PS1 mouse is shown at higher power ( h ) and illustrates diminishment of tracer accumulation below PAS abutting plaques, with brisk fluorescence in secondary penetrating arterioles, possibly representative of regurgitant flow. The distribution of MeX04 (amyloid-β plaque) label according to MPI is summarized in ( i ). TxRd tracer accumulation (MPI) is summarized in plaque-positive (>1 PAS abutting plaques) versus plaque-negative PAS in ( j ), showing marked diminishment of tracer accumulation that may be indicative of decreased flow; unpaired t -test. Correlation of MeX04 with ERTR7 pixel are is shown in ( k ); simple linear regression with 95% CI. Correlation of tracer MPI with ERTR7 label is shown in ( l ); simple linear regression with 95% CI. Correlation of tracer MPI with MeX04 MPI is shown in ( m ); simple linear regression with 95% CI. Cyan/CY5, MeX04; blue, lectin; green/FITC, ERTR7; red/Texas Red, TxRd. Scale bars = ( a ) 500 μm; ( h ) 50 μm. Data are depicted 30 min following intracisternal tracer infusion. Young: n = 141 vessels from 3 mice; Old: n = 139 vessels from 3 mice; APP/PS1: n = 141 vessels from 3 mice. Source data are provided as a Source Data file.

Article Snippet: Samples were permeabilized in 0.1% Triton X-100 with 5% goat serum (Sigma) for 2 h and then incubated for two days at 4 °C with a monoclonal rat ERTR7 antibody (NB100-64932, Novus Biologicals, Centennial CO, 1:1200).

Techniques: Fluorescence

Journal: Nature Communications

Article Title: Periarteriolar spaces modulate cerebrospinal fluid transport into brain and demonstrate altered morphology in aging and Alzheimer’s disease

doi: 10.1038/s41467-022-31257-9

Figure Lengend Snippet: At sites of arteriolar penetration ( a ), the intimal pia and epipia merge and distinct patterns of tracer deposition are observed around ensheathed vessels (i.e., Type A-D PAS), with free tracer passage, stagnation of the tracer bolus (open arrow) and/or cellular sequestration of tracer (solid arrows) noted. Cross and oblique PAS sections ( b , lower panel) depict tracer-positive cells studding the ERTR7-positive PAS cell network. As demonstrated in the oblique section and lower panel representing an enlargement of the boxed area, the TxRd tracer positive cells represent ED1-positive macrophages. ED1-positive cells are shown within Type B PAS of young, old, and APP/PS1 mice ( c ) and the distribution of tracer among ED1-positive macrophages in type A–D PAS of young mice is shown in ( d ). The number of ED1-expressing cells is variable among PAS types, being prominent in type D PAS ( e ) and correlating strongly with ERTR7 density ( f ); n = 107 vessels. Two-way ANOVA with Tukey’s multiple comparisons test in D. Simple linear regression with 95% CI in E, F; P values in legend refer to testing between slopes. Moreover, the depths of ED1-positive macrophages in PAS correlate strongly with pial (i.e., ERTR7) depth ( g ). The distribution of tracer among ED1-positive macrophages in type A–D PAS is quantified in ( h ). Two-way ANOVA with Tukey’s multiple comparisons test in ( h ). a – g Red/Texas Red, TxRd; green/FITC, ERTR7; white/CY5, ED1; blue, DAPI; Scale bars = ( a , upper panel) 30 μm; ( a , lower panels, b upper panel, c , g ) 10 μm; ( b , lower panel) 5 µm. Young: n = 87 vessels from 3 mice; Old: n = 71 vessels from 3 mice; APP/PS1: n = 66 vessels from 3 mice. Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Samples were permeabilized in 0.1% Triton X-100 with 5% goat serum (Sigma) for 2 h and then incubated for two days at 4 °C with a monoclonal rat ERTR7 antibody (NB100-64932, Novus Biologicals, Centennial CO, 1:1200).

Techniques: Expressing

Journal: OncoTargets and Therapy

Article Title:

CD26 as a Promising Biomarker for Predicting Prognosis in Patients with Pancreatic Tumors

doi: 10.2147/ott.s278736

Figure Lengend Snippet: Figure 2 CD26 expression in paratumoral tissues, benign pancreatic tumors, low- grade malignant pancreatic tumors and high-grade malignant pancreatic tumors. * represented P<0.05, *** represented P <0.001.

Article Snippet: After washing in PBS three times, the sections were blocked with goat serum (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) at 37°C for 1 h, followed by incubation with primary rabbit polyclonal anti-DPPIV antibodies (1:1200; cat. no. NB100-59021; Novus Biologicals, USA) overnight at 4° C. Unconjugated primary antibody was then removed by washing in PBS, and ready-to-use EnVision reagent (EnVision Detection System Peroxidase/DAB, Rabbit/ Mouse; Dako, Denmark) was used to bind the primary antibody.

Techniques: Expressing

Journal: OncoTargets and Therapy

Article Title:

CD26 as a Promising Biomarker for Predicting Prognosis in Patients with Pancreatic Tumors

doi: 10.2147/ott.s278736

Figure Lengend Snippet: Figure 1 Typical cytoplasmic staining of CD26 in (A) PDAC; (B) SPT; (C) NEN; (D) paratumoral pancreatic tissue; (E) IPMN; (F) serous neoplasms; (G) pancreatitis; (H) acinar cell neoplasm; (I) secondary pancreatic metastasis from oviduct serous adenocarcinoma and (J) MCN. The upper ones of each figure were ×100, and the lower ones were ×200.

Article Snippet: After washing in PBS three times, the sections were blocked with goat serum (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) at 37°C for 1 h, followed by incubation with primary rabbit polyclonal anti-DPPIV antibodies (1:1200; cat. no. NB100-59021; Novus Biologicals, USA) overnight at 4° C. Unconjugated primary antibody was then removed by washing in PBS, and ready-to-use EnVision reagent (EnVision Detection System Peroxidase/DAB, Rabbit/ Mouse; Dako, Denmark) was used to bind the primary antibody.

Techniques: Staining

Journal: OncoTargets and Therapy

Article Title:

CD26 as a Promising Biomarker for Predicting Prognosis in Patients with Pancreatic Tumors

doi: 10.2147/ott.s278736

Figure Lengend Snippet: Figure 3 Higher CD26 expression was associated with poorer DFS and OS in malignant pancreatic tumor patients. According to CD26 expression level, all the malignant pancreatic tumor patients were divided into high expression group and low expression group. Kaplan-Meier survival analysis and Log rank test showed that patients with lower CD26 expression had a better (A) disease-free survival and (B) overall survival than those with higher CD26 expression (P=0.048 and P=0.024).

Article Snippet: After washing in PBS three times, the sections were blocked with goat serum (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) at 37°C for 1 h, followed by incubation with primary rabbit polyclonal anti-DPPIV antibodies (1:1200; cat. no. NB100-59021; Novus Biologicals, USA) overnight at 4° C. Unconjugated primary antibody was then removed by washing in PBS, and ready-to-use EnVision reagent (EnVision Detection System Peroxidase/DAB, Rabbit/ Mouse; Dako, Denmark) was used to bind the primary antibody.

Techniques: Expressing