Journal: Frontiers in Molecular Neuroscience

Article Title: Ptbp2 re-expression rescues axon growth defects in Smn-deficient motoneurons

doi: 10.3389/fnmol.2024.1393779

Figure Lengend Snippet: Ptbp2 interacts with Smn in motoneurons. (A) Representative images of Smn-Ptbp2 PLA signal in cultured motoneurons at DIV 6 using anti-Smn and anti-Ptbp2 antibodies. Motoneuron morphology was visualized with anti-Tubb3 antibody. Scale bars, 10 and 5 μm (magnified areas). (B) Co-immunoprecipitation of Smn by anti-Ptbp2 from motoneuron lysate pre-treated with RNase A as indicated. (C) Representative images showing Smn immunofluorescence and Hnrnpr FISH in cultured motoneurons at DIV 6. Arrowheads indicate colocalization of Smn and Hnrnpr in granules. Scale bars, 10 and 2 μm (magnified areas). (D) Fluorescence intensity profiles of Smn and Hnrnpr at the location indicated by arrow 4 in (C) . (E) Representative images showing Ptbp2 and Smn immunofluorescence and Hnrnpr FISH in cultured motoneurons at DIV 6. Scale bars, 10 and 2 μm (magnified areas). (F) Fluorescence intensity profiles of Ptbp2, Smn and Hnrnpr at the location indicated by a line in (E) .

Article Snippet: Cells were fixed again for 10 min at room temperature in PLP, washed with DPBS, and stained with FITC-conjugated anti-Tubb3 antibody (130-131-158, Miltenyi Biotec).

Techniques: Cell Culture, Immunoprecipitation, Immunofluorescence, Fluorescence

Journal: Scientific Reports

Article Title: The Role of Transthyretin in Oligodendrocyte Development

doi: 10.1038/s41598-020-60699-8

Figure Lengend Snippet: TTR influences OPC differentiation. Representative images of neurospheres in 3D culture, isolated from SVZ derived NSCs of P21 ( A ) wild type and ( B ) TTR null mice. Scale bar 5 mm. ( C ) Quantitation of the potency of the NSC colonies isolated from the SVZ of P21 mice by neural colony-forming cell assays. There was a significant increase in the number of colonies with an average diameter ≥ 2 mm that were isolated from the SVZ of P21 TTR null mice when compared with wild type at the same age. Increases corresponded with a significant decrease in the number of colonies with an average diameter < 2 mm that were isolated from TTR null mouse SVZ compared to wild type. The colonies ≥ 2 mm in diameter are “NSC derived” and have self-renewal and multi-potential capabilities. Colonies < 2 mm diameter are “progenitor derived”. All data were expressed as the mean ± SEM. Statistical comparisons were performed using a one-way ANOVA with post hoc analysis using Sidak’s test as appropriate (data derived from n = 4 independent experiments per genotype and P < 0.05 was considered statistically significant). ( D ) Characterization of NSCs isolated from the SVZ of P21 TTR null mice and differentiated into the three neural lineages: oligodendrocytes, astrocytes and neurons. Nuclei were stained with DAPI; oligodendrocytes were stained with anti-Olig2; neurons were stained with anti-β-III-tubulin; astrocytes were stained with anti-GFAP. Lack of TTR promotes NSCs to differentiate into glial precursor cells. Differentiation assay for NSCs isolated from the SVZ of P21 TTR null mice showed a greater proportion of cells differentiating into a glial lineage, whereas the equivalent cells from wild type mice had a greater proportion differentiating into a neuronal lineage. All data were expressed as the mean ± SEM. Statistical comparisons were performed using a one-way ANOVA with post hoc analysis using Sidak’s test as appropriate (n = 3 independent experiments and P < 0.05 was considered statistically significant). (Modified from ).

Article Snippet: Cell pellets were collected and incubated with primary antibodies diluted in blocking solution: mouse anti-Olig2 (1:200; Merck Millipore, MAPN50), mouse anti-beta III tubulin (1:150; Merck Millipore, MAB1637), mouse anti-GFAP (1:200; Merck Millipore, MAB360), rabbit anti-TTR (1:300;; ABBIOTC, 250892) and incubated overnight at 4 °C.

Techniques: Isolation, Derivative Assay, Quantitation Assay, Staining, Differentiation Assay, Modification

Journal: Communications Biology

Article Title: Precise CAG repeat contraction in a Huntington’s Disease mouse model is enabled by gene editing with SpCas9-NG

doi: 10.1038/s42003-021-02304-w

Figure Lengend Snippet: a The procedure of neuronal differentiation of ES cells. For in vitro differentiation assay, we used ES clones transfected with prototype SpCas9-NG (not published), not ES clones described in Fig. . b Immunostaining of differentiated embryoid bodies. White arrows in the high magnified image of R6/2 clone #2 indicate HTT aggregates. All scale bars indicate 100 µm: the EB in genome-edited #2 (top) was almost twice larger than the others. c The differentiation scores based on a percentage of the β III tubulin staining in the circumference of embryoid bodies; 1: 0–25%; 2: 25–50%; 3: 50–75%; 4: 75–100%. Statistical analysis was performed using a two-tailed Mann–Whitney U-test ( p -value = 0.012).

Article Snippet: After three washes with PBS, the cells were incubated with Alexa Fluor 555 conjugated goat anti-mouse IgG (A21422, Thermo Fisher Scientific; 1:4000) at room temperature for 2 h. The cells were then incubated with a mouse anti-β III tubulin mouse antibody (T8660, Merck; 1:1000) at 4 °C overnight.

Techniques: In Vitro, Differentiation Assay, Clone Assay, Transfection, Immunostaining, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Cellular Neuroscience

Article Title: Alterations of protein composition along the rostro-caudal axis after spinal cord injury: proteomic, in vitro and in vivo analyses

doi: 10.3389/fncel.2014.00105

Figure Lengend Snippet: Quantification of DRG explant neurite outgrowth in response to culture media . M+GF (DMEM+ growth factors), M-GF (DMEM without growth factors), and conditioned media: CM CTR (control spinal cord), after SCI: CM RSCI (rostral segment), CM LSCI (lesioned segment), CM CSC (caudal segment) (A) . The image of TUJ1 stained DRG explants after selecting the area to be analyzed and thresholding the number of pixels covered by the extending neuritis, but not the DRG cell body (asterisk) (B) . Representative immunofluorescence images of TUJ1 positive DRG explants exposed to CM RSCI (C) , CM LSCI (D) , and CM CSC (E) . Scale bars (B–E) = 400 μm.

Article Snippet: Briefly, DRGs were incubated in mouse anti-Neuron-specific class III beta-tubulin (TUJ1) (1:200; Merck) for 24 h at 4°C.

Techniques: Staining, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Generation of the First Human In Vitro Model for McArdle Disease Based on iPSC Technology

doi: 10.3390/ijms232213964

Figure Lengend Snippet: Primary and secondary antibodies used for the immunocytochemistry analysis of the markers for ectoderm (Tuj1), endoderm (AFP) and mesoderm (SMA) after the in vitro spontaneous differentiation assay.

Article Snippet: Mouse anti-β tubulin isotype III (ectoderm) , 1:300 , Merck, Darmstadt, Germany; #T8660.

Techniques: Immunocytochemistry, In Vitro

Journal: Molecular neurobiology

Article Title: Microtubule-Actin Crosslinking Factor 1 is required for dendritic arborization and axon outgrowth in the developing brain

doi: 10.1007/s12035-015-9508-4

Figure Lengend Snippet: (A) MACF1 expression in cortical neurons. Cortical neurons from E14.5 control (MACF1loxP/+; Nex-cre) and MACF1loxP/loxP; Nex-cre brains were cultured for 5 days, and MACF1 localization was examined using immunostaining. Arrows indicate an accumulation of MACF1 in dendritic tips (B) MACF1 deletion causes aberrant actin arrangement and localization. Polymerized actins were visualized by phalloidin staining. Top panels show the patterns of polymerized actins at neurite tips. Middel and bottom panels show polymerized actins accumulated in the cytosol. (C) The levels of polymerized actin (F-actin) intensity in the cell body and at the neurite tip were quantified using Image J (NIH) software. n = 21 cells from 3 independent cultures using 3 mice for each condition. Statistical significance was determined by two-tailed Student's t-test. ***p < 0.001. (D) MACF1-deleted neurons show abnormal microtubule arrangement at the neurite tip. (E) Immunoblotting was performed to measure the levels of α-tubulin or acetylated-tubulin using E14.5 control and MACF1loxP/loxP; Nex-cre brain lysates (top panel). The levels of each tubulin were quantified in the bottom panel. n = 3 blots using 3 different lysates from 3 mice for each condition. Statistical significance was determined by two-tailed Student's t-test. ***p < 0.001.

Article Snippet: The following primary antibodies were used: Chicken anti-GFP (Invitrogen), Rabbit anti-GFP (Invitrogen), mouse anti-β-III Tubulin (Phosphosolutions), rabbit anti-acetyl-α-tubulin (Cell Signaling), and mouse anti-β-tubulin (Upstate).

Techniques: Expressing, Cell Culture, Immunostaining, Staining, Software, Two Tailed Test, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Pre-clinical development of AP4B1 gene replacement therapy for hereditary spastic paraplegia type 47

doi: 10.1038/s44321-024-00148-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mouse-anti-beta-Tubulin III , Synaptic Systems , Cat# 302304.

Techniques: Control, Variant Assay, Plasmid Preparation, Western Blot, Sequencing, Protease Inhibitor, Lysis, Gel Extraction, SYBR Green Assay, Enzyme-linked Immunospot