prolong gold antifade with dapi reagent p36935  (Thermo Fisher)

Journal: Brain Communications

Article Title: Downregulation of NEAT1 due to loss of TDP-43 function exacerbates motor neuron degeneration in amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcaf261

Figure Lengend Snippet: NEAT1 expression with or without TDP-43 pathology in ALS spinal motor neurons . ( A and B ) Postmortem spinal cords from patients with sporadic ALS and controls were thinned to 6 µm and stained via in situ hybridisation with NEAT1 -total specific probe ( A ) or NEAT1_2 specific probe ( B ) followed by double-immunostaining with anti-TDP-43 and DAPI to nuclear. The solid and dotted lines indicate the neuronal cell bodies and the nuclear, respectively. Scale bar: 10 µm. Asterisks: lipofuscin. (C and D) The signal intensities of NEAT1 -total ( C ) and NEAT1_2 ( D ) in spinal motoneurons in the control group and in groups with and without nuclear TDP-43 (nTDP-43+ and nTDP-43−, respectively) in ALS were measured. Each individual data point represents intensity of signal intensity per a nuclear for a single motor neuron. The data bars are expressed as mean ± SEM. Data were collected from 8 ALS patients and 8 controls. Statistical analysis was conducted via one-way ANOVA, followed by Tukey's multiple comparisons test. *** P < 0.001, **** P < 0.0001. ( E ) Postmortem spinal cords from patients with sporadic ALS and controls were stained via in situ hybridisation with NEAT1_2 negative control probe (magenta) followed by double-immunostaining with anti-TDP-43 and DAPI to nuclear. ( F ) In situ hybridisation ( NEAT1_2 ) and immunofluorescence images (NONO and SFPQ) of spinal motor neurons. Scale bar: 10 µm. A.U., arbitrary unit.

Article Snippet: The next day, the cells were incubated with secondary antibodies for 1 h and then encapsulated with ProLong Gold Antifade Reagent with DAPI ( P36935 ; Thermo Fisher Scientific).

Techniques: Expressing, Staining, In Situ, Hybridization, Double Immunostaining, Control, Negative Control, Immunofluorescence

Journal: Brain Communications

Article Title: Downregulation of NEAT1 due to loss of TDP-43 function exacerbates motor neuron degeneration in amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcaf261

Figure Lengend Snippet: Effect of TDP-43 depletion on NEAT1 expression . ( A ) Immunoblots of Neuro2a cell lysates treated with control siRNA (siCO) or each two siTDP-43 (#1 or #2). See for uncropped blots. ( B ) Quantification of the mRNA expression levels of Neat1 -total and Neat1_2 using cells in A ( n = 3 for each group). ( C ) Upper panels show in situ hybridisation ( Neat1_2 ) of Neuro2 treated with siCO or siTDP-43, and under panels show immunostaining (TDP-43). Scale bar: Right = 10 µm, Left = 100 µm. ( D ) Number of Neat1_2 signals per a Neuro2a cell ( n = 162 for siCO group, n = 169 for siTDP-43 group). ( E ) Immunoblots of Neuro2a cell lysates expressing FLAG-siRNA-resistant-TDP-43 and control siRNA or siTDP-43. In the TDP-43 immunoblot lane, the upper and lower bands indicate transfected TDP-43 (FLAG-resistant) and endogenous TDP-43, respectively. See for uncropped blots. ( F ) Quantification of the mRNA expression levels of Neat1 -total and Neat1_2 using cells in E ( n = 3 for each group). ( G ) Immunoblots of whole tissue lysate derived from primary cortical neurons with TDP-43 knockdown by EGFP-Cre lentivirus. See for uncropped blots. ( H ) Quantification of the mRNA expression levels of Neat1 -total and Neat1_2 using tissue lysate in G ( n = 3 for each group). ( I ) In situ hybridisation ( Neat1 -total and Neat1_2 ) and immunofluorescence images (TDP-43, ChAT and DAPI) of spinal motoneurons in TDP-43-cKO mice. The dotted lines indicate the nuclear. Scale bar: 10 µm. ( J ) Signal counts of Neat1 -total and Neat1_2 in the nuclei of spinal motoneurons ( n = 9–15 for each group). In B, F, and H, each individual data point represents the average of three measurement for a culture well. In D, each individual data point represents signal counts per a Neuro2a cell. In J, each individual data point represents signal counts per nuclear for a motor. The data bars are expressed as mean ± SEM. In B and F, statistical analysis was conducted via one-way ANOVA followed by Tukey's multiple comparisons test. In D, H and J, statistical analysis was conducted using unpaired t -test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. KD = knockdown.

Article Snippet: The next day, the cells were incubated with secondary antibodies for 1 h and then encapsulated with ProLong Gold Antifade Reagent with DAPI ( P36935 ; Thermo Fisher Scientific).

Techniques: Expressing, Western Blot, Control, In Situ, Hybridization, Immunostaining, Transfection, Derivative Assay, Knockdown, Immunofluorescence

Journal: Nature Communications

Article Title: NvPrdm14d -expressing neural progenitor cells contribute to non-ectodermal neurogenesis in Nematostella vectensis

doi: 10.1038/s41467-023-39789-4

Figure Lengend Snippet: A – C , E – G and I – K Confocal images of double fluorescence in situ hybridization for NvPrdm14d (green) and different neural marker genes (magenta). B , C Enlargements of NvPrdm14d + cells co-expressing (or not) NvSoxB(2) . D Quantification of NvPrdm14d + cells co-expressing NvSoxB(2) : 15–76% (average 50.5%, n = 7) of NvPrdm14d + cells co-express NvSoxB(2) . (F-G) Enlargements of NvPrdm14d + cells co-expressing (or not) NvElav1 . H Quantification of NvPrdm14d + cells co-expressing NvElav1 : 41–56% (average 50.2%, n = 4) of NvPrdm14d + cells co-express NvElav1 . J , K Enlargements of NvPrdm14d + cells co-expressing (or not) NvPOU4 . L Quantification of NvPrdm14d + cells co-expressing NvPOU4 : 50%–55% (average 52.8%, n = 3) of NvPrdm14d + cells co-express NvPOU4 . M – O Confocal images of fluorescence in situ hybridization for NvPrdm14d combined with the fluorescent labeling of proliferating cells by nuclear EdU staining, respectively shown in green and magenta. N – O Enlargements showing that some NvPrdm14d + cells are co-labeled by EdU. DAPI is shown in white in ( N″, O″ ) and in blue in the merged images ( N‴ , O‴ ). P Quantification of NvPrdm14d -expressing cells co-labeled by EdU. For 30 min pulse: 9–26% (average 18%, n = 8); for 2 h pulse: 22–33% (average 30%, n = 8) of NvPrdm14d + cells are EdU positive. Q Schematics summarizing the temporal expression of NvPrdm14d . The expression starts in a subset of endodermal neural progenitor cells that are still dividing and express NvSoxB(2) . The expression of NvPrdm14d continues in neurons expressing differentiation markers such as NvElav1 and NvPOU4 . Yellow arrows indicate co-labeled cells, while white arrows indicate cells with a single label. Embryos are counterstained for DNA in blue, the oral pole is oriented to the right, scale bars: 50 µm for full embryos ( A , E , I , M ), 10 µm for enlargements ( B , C , F , G , J , K , N , O ).

Article Snippet: Post-antibody washes and the TSA reactions were repeated as for the first probe; samples were then washed in TNTx, in PBTx, incubated with Hoechst 33342 [Thermo Fisher Scientific, 62249 (1:1000)] in PBTx and mounted in ProLong TM Gold antifade reagent with DAPI (Thermo Fisher Scientific, P36935).

Techniques: Fluorescence, In Situ Hybridization, Marker, Expressing, Labeling, Staining

Journal: Nature Communications

Article Title: NvPrdm14d -expressing neural progenitor cells contribute to non-ectodermal neurogenesis in Nematostella vectensis

doi: 10.1038/s41467-023-39789-4

Figure Lengend Snippet: Overview of the different types of cells labeled by NvPrdm14d ::GFP, shown in green. A NvPrdm14d: :GFP labels dividing endodermal neural progenitor cells (NPCs) as these cells are co-labeled by NvSoxB(2) ::mOrange, shown in magenta. Arrows indicate sister chromatids at anaphase. B Consistent with the expression pattern of NvPrdm14d , some rare ectodermal sensory neurons are labeled by NvPrdm14d ::GFP in the central region of the oral-aboral axis. Arrows indicate the sensory cilium; the white bracket indicates the ectoderm. C , D The endodermal neurons labeled by NvPrdm14d ::GFP are ganglion cells with a variable number of neurites, e.g. bi- and tripolar neurons as depicted here. No asymmetric distribution of these ganglion neurons was observed along the embryo axes. Arrows indicate neural somata. DAPI is shown in blue in the merged images. Scale bars: 10 µm.

Article Snippet: Post-antibody washes and the TSA reactions were repeated as for the first probe; samples were then washed in TNTx, in PBTx, incubated with Hoechst 33342 [Thermo Fisher Scientific, 62249 (1:1000)] in PBTx and mounted in ProLong TM Gold antifade reagent with DAPI (Thermo Fisher Scientific, P36935).

Techniques: Labeling, Expressing