Journal: eLife

Article Title: Lineage-specific intersection of endothelin and GDNF signaling in enteric nervous system development

doi: 10.7554/eLife.96424

Figure Lengend Snippet: ( a–d ) Wholemount preparations of E12.5 guts isolated from Pax2Cre/Ret/Rosa26 lacZ ( b ), Wnt1Cre/Ret/Rosa26 lacZ ( d ) mutant embryos and their littermate controls (a and c, respectively) stained with Xgal (blue). Arrows denote the wavefronts of lineage-labeled cells in the hindguts of the controls. Asterisks denote halted migration of Ret -deficient Pax2Cre -labeled cells at the cecum ( b ) and of Ret -deficient Wnt1Cre -labeled cells at the caudal end of the stomach ( d ). ( e–h ) Wholemount preparations of E12.5 guts isolated from Pax2Cre/Ret/Rosa26 td-Tomato ( f ), Wnt1Cre/Ret/Rosa26 td-Tomato ( h ) mutant embryos and their littermate controls (e and g, respectively) stained for neuronal 2H3 (green). Arrows denote the wavefronts of lineage-labeled cells in the hindguts of the control embryos. Arrowheads denote halted migration of Ret -deficient Pax2Cre -labeled cells at the cecum ( f ). ( i–l ) Wholemount preparations of E10.5 guts isolated from Pax2Cre/Ret/Rosa26 nT-nG ( j ), Wnt1Cre/Ret/Rosa26 nT-nG ( l ) mutant embryos and their littermate controls (i and k, respectively) stained for nuclear-GFP (green) and ENS progenitor marker p75 (magenta). Arrows denote the wavefronts of lineage-labeled cells in the midguts. Arrowheads denote p75 + Pax2Cre -labeled cells at the midgut areas ( i, j ). Abbreviations: es, esophagus; st, stomach. Scale bars, 500 μm ( a–d ), 250 μm ( e–h ), 50 μm ( i–l ).

Article Snippet: Antibodies used were rabbit anti-Pax2 (1:50, Invitrogen 08-1483), rabbit anti-AP2 (1:1000, Abcam ab52222), goat anti-Sox10 (1:200, Santa Cruz sc-17342), mouse anti-2H3 (1:100, DSHB), chicken anti-GFP (1:3000, Abcam ab13970), goat anti-p75 (1:300, R&D Systems AF1157), mouse anti-HuD (1:500, Santa Cruz sc-13577), rabbit anti-BLBP (1:500, Abcam ab32423), mouse anti-Tuj1 (1:1000, BioLegend MMS-435P), rabbit anti-CGRP (1:500-1000, Millipore PC205L), rabbit anti-NOS1 (1:500, Santa Cruz sc-648), goat anti-Ret (1:300, R&D Systems AF482), rabbit anti-phospho-Ret (Tyr1015) (1:300, Themo Fisher PA5-105930), and rabbit anti-phospho-Ret (Tyr1096) (1:300, Themo Fisher PA5-105796).

Techniques: Isolation, Mutagenesis, Staining, Labeling, Migration, Control, Marker

Journal: eLife

Article Title: Lineage-specific intersection of endothelin and GDNF signaling in enteric nervous system development

doi: 10.7554/eLife.96424

Figure Lengend Snippet: ( a–f ) Confocal z-stack images of 200 mm midgut sections isolated from E10.5 Pax2Cre/Rosa26 td-Tomato ( a–c ) and Wnt1Cre/Rosa26 td-Tomato embryos cultured for 3 days in the presence of GDNF ( a ), EDN3 ( b ), or NGF ( c ) and stained for neuronal Hu (cyan) and ENS progenitor marker p75 (yellow). Scale bar, 250 mm. ( g, h ) Compiled representations of the total area of outgrowth (Tomato + area around the explanted gut tube) from Pax2Cre/Rosa26 td-Tomato and Wnt1Cre/Rosa26 td-Tomato midgut slice explants in the presence of GDNF (black), EDN3 (orange), or NGF (blue). The data from GDNF-treated groups here are shown as the controls in . See for rostrocaudal orientation of these midgut slice preparations. Each dot represents one gut slice isolated from the indicated axial level; lost sections were not included as data points. The images shown in a–f represent sections that are 800–1000 μm caudal to the foregut-midgut junction (red boxes in g and h). p-Values: *p<0.05, **p<0.01, ***p<0.001, ns = not significant.

Article Snippet: Antibodies used were rabbit anti-Pax2 (1:50, Invitrogen 08-1483), rabbit anti-AP2 (1:1000, Abcam ab52222), goat anti-Sox10 (1:200, Santa Cruz sc-17342), mouse anti-2H3 (1:100, DSHB), chicken anti-GFP (1:3000, Abcam ab13970), goat anti-p75 (1:300, R&D Systems AF1157), mouse anti-HuD (1:500, Santa Cruz sc-13577), rabbit anti-BLBP (1:500, Abcam ab32423), mouse anti-Tuj1 (1:1000, BioLegend MMS-435P), rabbit anti-CGRP (1:500-1000, Millipore PC205L), rabbit anti-NOS1 (1:500, Santa Cruz sc-648), goat anti-Ret (1:300, R&D Systems AF482), rabbit anti-phospho-Ret (Tyr1015) (1:300, Themo Fisher PA5-105930), and rabbit anti-phospho-Ret (Tyr1096) (1:300, Themo Fisher PA5-105796).

Techniques: Isolation, Cell Culture, Staining, Marker

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: Images and quantitation of cholinotrophic basal forebrain neuron pathology within the nucleus basalis of Meynert in DSD+, DSD- and AMC cases (A–N) Photomicrographs show p75 NTR (brown, A-C), ChAT (purple, D-F) positive cells in AMC, DSD-, and DSD+ cases. Note the decrease in the number and intensity of both p75 NTR and ChAT labeled neurons in DSD- cases compared to an even greater reduction in DSD+ and globose shaped cells (black arrows) in DSD+ cases (C, F). Arrows in (A–F) mark cells shown at a higher magnification in the boxed areas located at the lower right corner of each panel. Dark brown AT8 (G, I) and TauC3 (H, J) bearing neurofibrillary tangles (NFTs) were observed only in the DSD- and DSD+ cases. Black arrows indicate globose shaped NFTs (G, H, and I) that are shown at a higher magnification in boxed areas adjacent to the lower magnification images. Note that not all neurons within the nbM (thin black arrows) contained tau pathology (G, H) in DSD- cases. Moreover, TauC3 staining revealed two NFT phenotypes that displayed either peripherally located or intense labeling that filled the entire structure (see boxed images adjacent to (H) and (J)). Scale bar in F = 50 μm and inset = 20 μm applies to panels A-E. Scale bar in J = 20 μm and inset = 20 μm applies to (G–J). Histograms show a significant reduction in both p75 NTR (K) and ChAT (L) positive cells in DSD+ compared to AMC. Although no significant were found in number of AT8 or TauC3 NFT positive cells (M), there was an increase in NTs in DSD+ compared to DSD- (N). ACM n = 5, DSD- n = 5, DSD+ n = 10. Data shown are presented as mean ± SEM. Statistical significance was determined using the Kruskal–Wallis’s test followed by Dunn’s test for comparisons across three clinical groups, and the Mann–Whitney test for comparisons between two groups (DSD- vs. DSD+). Significance levels (∗) were set at: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Antibodies were added to blocking buffer and membranes incubated overnight (4°C) in mouse anti-p75 NTR ([1:150], Santa Cruz Biotechnology Cat# sc-271708, RRID: AB_10714958 ), rabbit anti-ChAT ([1:500] (Proteintech Cat# 20747-1-AP, RRID: AB_10898169 ), rabbit anti-TrkA ([1:150], Fitzgerald Industries International Cat# 20R-TR013, RRID: AB_1289098 ) or rabbit anti-proNGF ([1:250], Alomone Labs Cat# ANT-005, RRID: AB_2040021 ), washed, incubated with horseradish peroxidase-conjugated with either a goat anti-mouse IgG (1:200) or goat anti-rabbit IgG (1:200) secondary antibody at RT, visualized by chemiluminescence (Kodak Image Station 440CF; Perkin-Elmer, Wellesley, MA), and quantified with Kodak 1.

Techniques: Quantitation Assay, Labeling, Staining, MANN-WHITNEY

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: Immunofluorescent images and quantification of p75 NTR neurons with and without AT8 and MAP2, as well as Thioflavin S single and AT8/Thioflavin S dual-labeled nbM cells in DSD+ and DSD-cases. (A–U) Immunofluorescent neurons labeled with p75 NTR (A, E, green), MAP2 (B, F, red), AT8 (C, G, Cyan), ThS (blue) (I, L) and merge images (D, H, K, N, R) in DSD- and DSD+ cases. In the DSD-cases there were greater p75 NTR cells AT8 negative neurons and p75 NTR MAP2 dual labeled neurons (A-D) compared to the DSD+ cases (E–H). Note that not all p75 NTR cells colocalize with AT8 or MAP2 in both DS groups (D, H). To determine the stage of a tangle tissue was stained for ThS, a marker of advanced pathology and the early stage AT8 phosphorylation antibody. Note that there are only a few ThS-labeled tangles that displayed AT8 in both DS groups (yellow arrows), compared to single ThS tangles, which were greater in DSD+ than in DSD- (white arrows) (I-L). Note that ThS positive NFTs that do not contain AT8 also do not colocalize with MAP2 (O-R). Scale bar in F = 25 μm applies to panels A-G, N = 25 μm applies to panels I-M and R = 10 μm and applies to O-Q. Graph showing a significant reduction in both p75 NTR AT8 immuno-negative, and p75 NTR MAP2 dual labeled neurons in DSD+ compared to DSD- (S). ThS-positive cells were greater (T), while the percentage of double-labeled cells with AT8 and ThS decreased (U) in DSD+ compared to individuals without dementia. DSD- n = 5, DSD+ n = 5. Data are presented as mean ± SEM. Statistical significance was determined using Mann–Whitney test for comparisons between DSD- and DSD+. Significance levels (∗) were set at: ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Antibodies were added to blocking buffer and membranes incubated overnight (4°C) in mouse anti-p75 NTR ([1:150], Santa Cruz Biotechnology Cat# sc-271708, RRID: AB_10714958 ), rabbit anti-ChAT ([1:500] (Proteintech Cat# 20747-1-AP, RRID: AB_10898169 ), rabbit anti-TrkA ([1:150], Fitzgerald Industries International Cat# 20R-TR013, RRID: AB_1289098 ) or rabbit anti-proNGF ([1:250], Alomone Labs Cat# ANT-005, RRID: AB_2040021 ), washed, incubated with horseradish peroxidase-conjugated with either a goat anti-mouse IgG (1:200) or goat anti-rabbit IgG (1:200) secondary antibody at RT, visualized by chemiluminescence (Kodak Image Station 440CF; Perkin-Elmer, Wellesley, MA), and quantified with Kodak 1.

Techniques: Labeling, Staining, Marker, Phospho-proteomics, MANN-WHITNEY

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: FC cholinotrophic protein levels in AMC, DSD- and DSD+ (A–D) Representative immunoblots, and bar graphs show a significant upregulation of (A) proNGF and (B) p75 NTR , while (C) ChAT protein was downregulated between AMC and DSD+. (D) TrkA protein levels were stable across the groups analyzed. ACM n = 5, DSD- n = 5, DSD+ n = 13. Data are presented as mean ± SEM. Statistical significance was determined using the Kruskal–Wallis’s test followed by Dunn’s test for comparisons across three clinical groups. Significance levels (∗) were set at: ∗ p < 0.05.

Article Snippet: Antibodies were added to blocking buffer and membranes incubated overnight (4°C) in mouse anti-p75 NTR ([1:150], Santa Cruz Biotechnology Cat# sc-271708, RRID: AB_10714958 ), rabbit anti-ChAT ([1:500] (Proteintech Cat# 20747-1-AP, RRID: AB_10898169 ), rabbit anti-TrkA ([1:150], Fitzgerald Industries International Cat# 20R-TR013, RRID: AB_1289098 ) or rabbit anti-proNGF ([1:250], Alomone Labs Cat# ANT-005, RRID: AB_2040021 ), washed, incubated with horseradish peroxidase-conjugated with either a goat anti-mouse IgG (1:200) or goat anti-rabbit IgG (1:200) secondary antibody at RT, visualized by chemiluminescence (Kodak Image Station 440CF; Perkin-Elmer, Wellesley, MA), and quantified with Kodak 1.

Techniques: Western Blot

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: Summary of changes in the cholinotrophic basal forebrain connectome in DS Diagrammatic sagittal view of the human brain (A) and a modified stacked bar graph (B) illustrating differences in the pathobiology of the cholinotrophic projection system between non-trisomy age-matched control (AMC), DS without dementia (DSD-) and DS with dementia (DSD+) individuals with DS. Frontal cortex protein levels for ChAT (pink), proNGF (green), p75 NTR (purple) and TrkA (light blue). Nucleus basalis NFTs of ThS (blue), AT8 (orange) and TauC3 (black) and counts of p75 NTR and ChAT (red) neurons. Created with BioRender.com .

Article Snippet: Antibodies were added to blocking buffer and membranes incubated overnight (4°C) in mouse anti-p75 NTR ([1:150], Santa Cruz Biotechnology Cat# sc-271708, RRID: AB_10714958 ), rabbit anti-ChAT ([1:500] (Proteintech Cat# 20747-1-AP, RRID: AB_10898169 ), rabbit anti-TrkA ([1:150], Fitzgerald Industries International Cat# 20R-TR013, RRID: AB_1289098 ) or rabbit anti-proNGF ([1:250], Alomone Labs Cat# ANT-005, RRID: AB_2040021 ), washed, incubated with horseradish peroxidase-conjugated with either a goat anti-mouse IgG (1:200) or goat anti-rabbit IgG (1:200) secondary antibody at RT, visualized by chemiluminescence (Kodak Image Station 440CF; Perkin-Elmer, Wellesley, MA), and quantified with Kodak 1.

Techniques: Modification, Control

Journal: The EMBO Journal

Article Title: T-cell-derived IFN-γ suppresses T follicular helper cell differentiation and antibody responses

doi: 10.1038/s44318-025-00414-3

Figure Lengend Snippet: Reagents and tools table

Article Snippet: InVivoMab α-IL-12 blocking antibody , BioXcell , Clone R2-9A5 #BE0233.

Techniques: Transgenic Assay, Mouse Assay, Recombinant, Blocking Assay, Control, Staining, Sequencing, Expressing, Derivative Assay, Lysis, Software, Cell Isolation, Microscopy, Real-time Polymerase Chain Reaction

Journal: The EMBO Journal

Article Title: T-cell-derived IFN-γ suppresses T follicular helper cell differentiation and antibody responses

doi: 10.1038/s44318-025-00414-3

Figure Lengend Snippet: Reagents and tools table

Article Snippet: InVivoMab α-IL-12 blocking antibody , BioXcell , Clone R2-9A5 #BE0233.

Techniques: Transgenic Assay, Mouse Assay, Recombinant, Blocking Assay, Control, Staining, Sequencing, Expressing, Derivative Assay, Lysis, Software, Cell Isolation, Microscopy, Real-time Polymerase Chain Reaction