fluoromount-g with or without dapi 00-4959-52  (Thermo Fisher)

Journal: bioRxiv

Article Title: Ift43 Controls the Ciliary Levels of Gli2 and Gli3

doi: 10.64898/2026.01.14.699321

Figure Lengend Snippet: (A) Electroretinograms (ERGs) of control ( Ift43 flox/+ /iCre75) and experimental ( Ift43 flox/flox /iCre75) littermates at postnatal day 30 (P30). Each dot represents the ERG response from each eye of an animal. ****p<0.0001, ns not significant by unpaired t-tests. (B) Transmission electron microscopy images of retinal sections from control ( Ift43 flox/+ /iCre75) and experimental ( Ift43 flox/flox / iCre75) littermates at postnatal day 21 and 30 (P21 and P30). Scale bar = 2 µm ( Ba-Bc ), 1 µm ( Bd-Bf ), 2 µm ( Bg-Bi ), 1 µm ( Bj-Bl ). Arrows in Bd-f mark outer segments. Arrows in Bg-i mark inner segments and asterisks in Bi mark extracellular vesicles. Arrows in Bj-k mark examples of outer segment bases. Asterisk in Bl marks extracellular vesicles. OS: outer segment; IS: inner segment. n=3 animals examined by TEM. (C) High magnification TEM images of extracellular vesicles in the inner segment (arrow, Ca ) and vesicle (arrow, Cb ) in the Golgi complex (G) of experimental ( Ift43 flox/flox /iCre75) littermates at postnatal day 30. Scale bar = 0.5 μm ( Ca ), 1 μm ( Cb ). n=3 animals examined by TEM. (D) Confocal images of retinal cross sections (agarose embedded) at P25. Control ( Ift43 flox/+ /iCre75) and experimental ( Ift43 flox/flox /iCre75) littermate mice stained with antibodies against outer segment proteins (green). Rhodopsin clone 4D2 ( Da ), Red/Green opsin ( Db ), Wheat germ agglutinin (WGA, Dc ). Nuclei are labelled with DAPI (4′,6-diamidino-2-phenylindole) in blue. Images are maximum intensity z-projections of 20 images taken at 0.7 µm intervals. OS: outer segment; IS: inner segment; ONL: outer nuclear layer, OPL: outer plexiform layer; INL: inner nuclear layer. Scale bars = 20 µm. Protein intensity profiles (green) along white lines shown at right. Magenta arrows: outer segment proteins mislocalized to inner segment or outer nuclear layer. Magenta asterisks mark outer segment proteins mislocalized to outer nuclear layer. n=4 animals examined by immunofluorescence microscopy. Raw data underling the graphs are available in S5 Data.

Article Snippet: Samples were mounted as whole mounts in Fluoromount-G without DAPI (Southern Biotech, Birmingham AL USA, 0100-01) for imaging.

Techniques: Control, Transmission Assay, Electron Microscopy, Staining, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: IFT20 regulates lymphatic endothelial cell-cell junctions via endocytic trafficking of VE-cadherin

doi: 10.1101/2025.01.15.631989

Figure Lengend Snippet: (A) Ift20 was knocked out of immortalized mouse lymphatic endothelial cells (mLEC) using CRISPR/Cas9. Immunofluorescence micrographs confirm loss of IFT20 ( left panel ) and abrogation of primary cilia assembly in IFT20 KO cells after 24 h serum starvation in media containing 0.2% FBS ( right panel, quantified in B ). DAPI ( blue ). Scale bars = 10 μm. (B) Quantification of primary cilia incidence in parental and IFT20 KO mLECs from A . Data points represent each of three independent experiments, each quantifying 100+ cells. (C) Epifluorescence micrographs of parental and IFT20 KO mLECs with phalloidin-labeled F-actin ( left panel ) or ZO-1 immunofluorescence staining ( right panel ). DAPI ( blue ). Scale bars = 10 μm. (D) Chemotaxis potential of parental and IFT20 KO mLECs was assessed by transwell migration assay. Cells were seeded in the upper chamber in starving media containing 0.2% FBS. Lower wells were filled with media containing either 0.2% FBS, 10% FBS, or 500 ng/mL VEGF-C in 0.2% FBS. After 24 h migration, membranes were fixed, mounted in DAPI mounting media, and imaged via epifluorescence microscopy. Scale bar = 200 μm. (E) Quantification of transwell migration from D . Migration is graphed relative to 10% FBS-stimulated mLEC migration. Each data point represents an average from 5 FOVs per membrane. Quantification is representative of three independent experiments in which three membranes were quantified per experimental condition. (F) Ift20 was targeted by siRNA in primary human dermal lymphatic endothelial cells (HDLECs). F-actin was labelled with phalloidin, and ZO-1 and VE-cadherin were labeled by immunofluorescence. Micrographs are MIPs from laser scanning confocal microscopy. DAPI ( blue ). Scale bars F-actin and ZO-1 = 10 μm. Scale bar VECAD left panel = 50 μm, right panel = 10 μm. *p<0.05, **p<0.005.

Article Snippet: Samples were mounted in Fluoromount-G without DAPI (Fisher Scientific, OB100-01).

Techniques: CRISPR, Immunofluorescence, Labeling, Staining, Chemotaxis Assay, Transwell Migration Assay, Migration, Epifluorescence Microscopy, Membrane, Confocal Microscopy

Journal: bioRxiv

Article Title: IFT20 regulates lymphatic endothelial cell-cell junctions via endocytic trafficking of VE-cadherin

doi: 10.1101/2025.01.15.631989

Figure Lengend Snippet: (A) Non-serum starved control HDLECs were immunostained for IFT20 ( green ) and RAB5 ( magenta ). Scale bars = 10 μm. (B) Control HDLECs were serum starved for 24 h in 1/5 EGM-2MV and 4/5 EBM-2 (hereafter, starving media) and then treated for 6 h with 2 μg/mL VEGF-C in starving media. Cells were then fixed or placed into EBM-2 basal media for 1.5 or 3 h washout. IFT20 ( green ) and RAB5 ( magenta ) were detected by immunofluorescence microscopy. (A-B) Micrographs are single 0.3 μm z-slices from laser scanning confocal microscopy. DAPI ( blue ). Scale bars = 10 μm. (C) Quantification of B . RAB5+ IFT20+ area is graphed as a percentage of total IFT20+ area per FOV representing one of two biological replicates. Quantification is representative of 100+ control and 100+ IFT20 KD HDLECs. ***p<0.001, ****p<0.0001.

Article Snippet: Samples were mounted in Fluoromount-G without DAPI (Fisher Scientific, OB100-01).

Techniques: Control, Immunofluorescence, Microscopy, Confocal Microscopy

Journal: bioRxiv

Article Title: IFT20 regulates lymphatic endothelial cell-cell junctions via endocytic trafficking of VE-cadherin

doi: 10.1101/2025.01.15.631989

Figure Lengend Snippet: (A) Control and IFT20 KD HDLECs were serum starved for 24 h in in 1/5 EGM-2MV and 4/5 EBM-2 (hereafter, starving media). Cells were then treated with 2 μg/mL VEGF-C for 6 h in starving media and fixed or placed in EBM-2 basal media for 1.5 or 3 h washout. VE-cadherin ( white ) was detected by immunofluorescence microscopy. Scale bar = 50 μm. (B) Quantification of granularity to measure breakdown in VE-cadherin cell-cell junctions from A . Each data point represents the average granularity of a single FOV normalized per cell from each of two biological replicates. *p<0.05, **p<0.005. (C) Control and IFT20 KD HDLECs were serum starved for 24 h in starving media. Cells were then treated with 2 μg/mL VEGF-C for 6 h in starving media and fixed or placed in EBM-2 basal media for 1.5 or 3 h washout. VE-cadherin ( green ) and RAB5 ( magenta ) were detected by immunofluorescence microscopy. Scale bar = 10 μm. (D) Quantification of VE-cadherin+ RAB5+ area from C . Graphs show one representative biological replicate of two, each comprising two technical replicates with 100+ cells per condition. In graphs depicting individual CTRL or KD data, data is graphed relative to control 24 h starve treatment (column 1). In the right graph where the two cell lines are combined, data is graphed as total area (px 2 ) for both datasets. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. (A, C) Micrographs are single 0.3 μm z-slices from laser scanning confocal microscopy. DAPI ( blue ).

Article Snippet: Samples were mounted in Fluoromount-G without DAPI (Fisher Scientific, OB100-01).

Techniques: Control, Immunofluorescence, Microscopy, Confocal Microscopy

Journal: bioRxiv

Article Title: A role for Lin28a in aging-associated decline of adult hippocampal neurogenesis

doi: 10.1101/2022.01.03.474756

Figure Lengend Snippet: (A) Experimental paradigm for testing pattern separation in mice. (B) The freezing of 2 mo and 10 mo mice in contexts A and B in test sessions 1 through 6. Young mice (2 mo) discriminated the two contexts in session 4, while aging mice (10 mo) showed discrimination in session 6. (2 mo mice n=10, 10 mo mice n=10; two-tailed paired t-test; session 1: 2 mo t 9 =0.1441, P=0.8886; 10 mo t 9 =1.609, P=0.1421; session 2: 2 mo t 9 =1.036, P=0.3272; 10 mo t 9 =0.2128, P=0.8362; session 3: 2 mo t 9 =1.391, P=0.1976; 10 mo t 9 =0.8787, P=0.4024; session 4: 2 mo t 9 =2.672, *P=0.0255; 10 mo t 9 =0.6441, P= 0.5356; session 5: 2 mo t 9 =6.572, ***P= 0.0001; 10 mo t 9 =2.220, P=0.0535; session 6: 2 mo t 9 =7.818, ****P<0.0001; 10 mo t 9 =3.897, **P=0.0036). (C) Diagram showing TAM or Veh was administered to Nestin-Cre ERT2 ::Lin28a flox/flox mice to knockout Lin28a from Nestin + NPCs. Mice were perfused four weeks later for examination. (D) Confocal images showing MCM2 + proliferating cells in the SGZ of Nestin-Cre ERT2 ::Lin28a flox/flox mice treated with Veh or TAM. Scale bar: 50 μm. (E) Knocking out Lin28a from Nestin + NPCs decreased the density of MCM2 + cells in the SGZ (Veh n=5 mice, TAM n=4 mice; two-tailed unpaired t-test, t 7 =3.638, **P=0.0083). (F) Knocking out Lin28a from Nestin + NPCs decreased the percentage of MCM2 + cells in total cells (DAPI + ) in the SGZ and GCL (Veh n=5 mice, TAM n=4 mice; two-tailed unpaired t-test, t 7 =3.116, *P=0.0158). (G) Confocal images showing DCX + cells in the DG of Nestin-Cre ERT2 ::Lin28a flox/flox mice treated with Veh or TAM. Scale bar: 50 μm. (H) Loss of Lin28a from Nestin + NPCs decreased the density of DCX + cells in the GCL (Veh n=5 mice, TAM n=5 mice; two-tailed unpaired t-test, t 8 =6.252, ***P=0.00023). (I) Loss of Lin28a from Nestin + NPCs decreased the percentage of DCX + cells in total cells (DAPI + ) in the SGZ and GCL (Veh n=5 mice, TAM n=5 mice; two-tailed unpaired t-test, t 8 =3.656, **P=0.0064). (J) Diagram showing TAM was administered to Nestin-Cre ERT2 ::Lin28a +/+ ::Ai14 or Nestin-Cre ERT2 ::Lin28a flox/flox ::Ai14 mice. Mice were perfused six weeks later for examination. (K) Confocal images showing dTomato + cells in the GCL of Nestin-Cre ERT2 :: Lin28a +/+ ::Ai14 (Ctrl) or Nestin-Cre ERT2 ::Lin28a flox/flox ::Ai14 (Lin28a f/f ) mice. Scale bar: 20 μm. (L) Loss of Lin28a from Nestin + NPCs decreased the density of dTomato + cells in the GCL (Ctrl n=5 mice, Lin28a f/f n=4 mice; two-tailed unpaired t-test, t 7 =3.113, *P=0.0170). (M) Loss of Lin28a from Nestin + NPCs decreased the percentage of dTomato + cells in total cells (DAPI + ) in the GCL (Ctrl n=5 mice, Lin28a f/f n=4 mice; two-tailed unpaired t-test, t 7 =4.780, **P=0.0020). (N) Schematics showing that Nestin-Cre ERT2 ::Lin28a flox/flox mice were treated with Veh (Ctrl) or TAM (Lin28a f/f ), followed by behavioral tests six weeks later. (O) The freezing of Veh- and TAM-treated mice in contexts A and B in test sessions 1 through 6. (Veh mice n=12, TAM mice n=10; two-tailed paired t-test; session 1: Veh t 11 =0.6717, P=0.5156; TAM t 9 =1.151, P=0.2795; session 2: Veh t 11 =0.8907, P=0.3921; TAM t 9 =1.177, P=0.2694; session 3: Veh t 11 =2.867, *P=0.0153; TAM t 9 =0.6078, P=0.5583; session 4: Veh t 11 =3.427, **P= 0.0057; TAM t 9 =1.925, P=0.0863; session 5: Veh t 11 =5.504, ***P= 0.0002; TAM t 9 =3.434, **P=0.0075; session 6: Veh t 11 =3.530, **P=0.0047; TAM t 9 =2.614, *P=0.0281).

Article Snippet: Brains were sliced into 60 μm sections, which were then mounted on slides with Fluoromount-G anti-fade medium without DAPI (SouthernBiotech).

Techniques: Two Tailed Test, Knock-Out

Journal: bioRxiv

Article Title: A role for Lin28a in aging-associated decline of adult hippocampal neurogenesis

doi: 10.1101/2022.01.03.474756

Figure Lengend Snippet: (A) Schematics showing lentiviral vectors expressing GFP or Wnt3a. (B) Schematics showing that lentiviral vectors were injected into the DG of adult C57 mice. Mice were perfused for examination one week later. (C) Confocal image showing infection of cells in the DG by lentivirus expressing Wnt3a-p2A-GFP. Scale bar: 50 μm. (D) Representative western blotting showing β-catenin expression level in the DG of animals injected with lentiviruses expressing GFP or Wnt3a. β-tubulin was used as internal control. (E) Relative expression level of β-catenin in the DG of animals injected with GFP or Wnt3a lentiviruses (GFP: n=8 mice per sample; Wnt3a: n=9 mice per sample; t 15 =2.7375, *P=0.0153). (F) RNAScope images showing colocalization of Lin28a mRNA with cell bodies of Sox2-expressing NPCs (indicated by Sox2 mRNA) in animals injected with GFP or Wnt3a lentiviruses. Dotted lines outline the cell bodies of interested cells. Insets in the merged images show the localization of Lin28a and Sox2 mRNA around the nucleus (DAPI) within the cell bodies. Scale bar: 5 μm. (G) Wnt3a increased the mRNA expression level of Lin28a in Sox2-expressing NPCs. (GFP: N=3 mice, n= 56 cells; Wnt3a: N=3 mice, n=62 cells; two-tailed unpaired t-test, t 116 =3.9875, ***P=0.0001). (H) Upper panel: Schematics showing FLEX lentiviral vectors expressing GFP (Ctrl) or GFP-p2A-Ctnnb1 (Ctnnb1). Lower panel: Schematics showing that FLEX lentiviral vectors expressing GFP or GFP-p2A-Ctnnb1 were injected into the DG of adult Nestin-Cre mice. Mice were perfused for examination one week later. (I) RNAScope images showing colocalization of Lin28a mRNA with cell bodies of GFP-labeled NPCs in the DG of Nestin-Cre mice injected with Cre-dependent lentiviral vectors expressing GFP or Ctnnb1. Scale bar: 5 μm. (J) Upregulation of Ctnnb1 increased the mRNA expression level of Lin28a in NPCs. (GFP: N=3 mice, n= 63 cells; Ctnnb1: N=3 mice, n=37 cells; two-tailed unpaired t-test, t 98 =13.57, ****P<0.0001).

Article Snippet: Brains were sliced into 60 μm sections, which were then mounted on slides with Fluoromount-G anti-fade medium without DAPI (SouthernBiotech).

Techniques: Expressing, Injection, Infection, Western Blot, Control, RNAscope, Two Tailed Test, Labeling

Journal: bioRxiv

Article Title: A role for Lin28a in aging-associated decline of adult hippocampal neurogenesis

doi: 10.1101/2022.01.03.474756

Figure Lengend Snippet: (A) Schematics showing that adult Nestin-Cre ERT2 ::Lin28a flox/flox mice (Lin28a f/f ) or Cre −/− litter mates (Ctrl) were treated with TAM, followed by injection of lentiviral vectors expressing GFP or Wnt3a in the DG, one week later. Four weeks after viral injection, the animals were perfused. (B) Confocal images showing DCX + newborn neurons in the DG of Ctrl and Lin28a f/f mice treated with TAM, and injected with GFP or Wnt3a lentiviruses. Scale bar: 20 μm. (C) Wnt3a increased the density of DCX + cell in the DG of Ctrl mice, but not in Lin28a f/f mice. (Ctrl: GFP n=4 mice, Wnt3a n=5 mice; two-tailed unpaired t-test, t 7 =2.554, *P=0.0379; Lin28a f/f : GFP n=6 mice, Wnt3a n=6 mice; two-tailed unpaired t-test, t10=0.3891, P=0.7054; Ctrl GFP vs. Lin28a f/f GFP: two-tailed unpaired t-test, t 8 =6.341, ***P=0.0002). (D) Wnt3a increased the percentage of DCX + cell in the total cells (DAPI + ) of the GCL in Ctrl mice, but not in Lin28a f/f mice. (Ctrl: GFP n=4 mice, Wnt3a n=5 mice; two-tailed unpaired t-test, t 8 =2.718, *P=0.0299; Lin28a f/f : GFP n=6 mice, Wnt3a n=6 mice; two-tailed unpaired t-test, t 10 =1.113, P=0.2918; Ctrl GFP vs. Lin28a f/f GFP: two-tailed unpaired t-test, t 8 =3.877, **P=0.0047). (E) Schematics showing that adult Nestin-Cre ERT2 ::Lin28a flox/flox ::Ai14 mice (Lin28a f/f ) or Nestin-Cre ERT2 ::Ai14 mice (Ctrl) were treated with TAM, followed by injection of lentiviral vectors expressing GFP or Wnt3a in the DG, one week later. Four weeks after viral injection, the animals were perfused for further analysis. (F) Confocal images showing dTomato + adult-born neurons in the DG of Ctrl and Lin28a f/f mice treated with TAM, and injected with GFP or Wnt3a lentiviruses. Scale bar: 20 μm. (G) Wnt3a increased the density of dTomato + cell in the DG of Ctrl mice, but not in Lin28a f/f mice. (Ctrl: GFP n=5 mice, Wnt3a n=5 mice; two-tailed unpaired t-test, t 8 =2.921, *P=0.0193; Lin28a f/f : GFP n=6 mice, Wnt3a n=6 mice; two-tailed unpaired t-test, t 10 =0.03851, P=0.9700; Ctrl GFP vs. Lin28a f/f GFP: two-tailed unpaired t-test, t 9 =2.705, *P=0.0242). (H) Wnt3a increased the percentage of dTomato + cell in the total cells (DAPI + ) of the GCL in Ctrl mice, but not in Lin28a f/f mice. (Ctrl: GFP n=5 mice, Wnt3a n=5 mice; two-tailed unpaired t-test, t 8 =3.531, **P=0.0077; Lin28a f/f : GFP n=6 mice, Wnt3a n=6 mice; two-tailed unpaired t-test, t 10 =0.5937, P=0.5659; Ctrl GFP vs. Lin28a f/f GFP: two-tailed unpaired t-test, t 9 =2.714, *P=0.0238). (I) Schematics showing lentiviral vectors expressing dTomato or dnWnt1 (upper panel), and FLEX lentiviral vectors expressing GFP (Ctrl) or GFP-p2A-Lin28a (Lin28a-OE) (lower panel). (J) Schematics showing that lentiviral vectors expressing dTomato or dnWnt1 were injected into the DG of adult Nestin-Cre mice, and Cre-dependent Ctrl or Lin28a-OE lentiviral vectors were injected 2 weeks later. Four more weeks later, the animals were perfused. (K) Confocal images showing DCX + newborn neurons in the DG of Ctrl and Lin28a f/f mice treated with TAM, and injected with GFP or Wnt3a lentiviruses. Scale bar: 20μm. (L) dnWnt1 decreased the percentage of DCX + cell in the total cells (DAPI + ) of the GCL, whereas upregulating Lin28a in NPCs recovered the number of DCX + cells. (dTomato: Ctrl n=4 mice, Lin28a-OE n=5 mice; two-tailed unpaired t-test, t 7 =3.993, **P=0.0052; dnWnt1: Ctrl n=4 mice, Lin28a-OE n=4 mice; two-tailed unpaired t-test, t 6 =6.199, ***P=0.0008; dTomato Ctrl vs. dnWnt1 Ctrl: two-tailed unpaired t-test, t 6 =4.421, **P=0.0045; dTomato Lin28a-OE vs. dnWnt1 Lin28a-OE: two-tailed unpaired t-test, t 7 =4.181, **P=0.0041).

Article Snippet: Brains were sliced into 60 μm sections, which were then mounted on slides with Fluoromount-G anti-fade medium without DAPI (SouthernBiotech).

Techniques: Injection, Expressing, Two Tailed Test