Microphot sa microscope

A Nikon-Microphot-SA microscope was used to capture images of DAB-stained embryos (40×). Images for the Sxl RNAi rescue experiment were acquired using a 100×, 1.49 NA Apo TIRF oil immersion objective on a Nikon Ti-E system fitted with a Yokagawa CSU-X1 spinning disk head, Hamamatsu Orca Flash 4.0 v2 digital CMOS camera, and Nikon LU-N4 solid state laser launch. Imaging for all other smFISH and fluorescent immunostaining experiments was performed on a Nikon A1 inverted laser-scanning confocal microscope.
Images were assembled using ImageJ (NIH) and Adobe Photoshop and Illustrator software to crop regions of interest, adjust brightness and contrast, generate maximum-intensity projections, and separate or merge channels. To assess the spreading of the RNAs or protein in different mutant backgrounds compared to the control we generated plot profiles using ImageJ. The posterior-most 75 µm of each embryo was plotted for comparison, and embryos from a single biological replicate are plotted in figures given that variation between fluorescence between replicates obscured the pole plasm distribution trends if embryos from all replicates were plotted together.

Leaves were harvested and fixed immediately in ethanol/glacial acetic acid (1:1) for 12h at 4 °C. After fixation, leaves were dehydrated in an ethanol series (50, 70, 80, 100% for 20min each). Subsequently, the leaves were immersed in a clearing solution (chloral hydrate/glycerol/H₂O (8:2:1). Samples were observed with a Nikon MicroPhot-SA microscope using DIC optics and images were captured with a Nikon CoolPix 990 digital camera. Six images per leaf were taken (i.e. three consecutive images per lamina side). Cell density was determined by counting all the cells included in a fixed image area (six images per leaf; five leaves per genotype). The total number of cells per leaf (referred to as cell number) was then calculated from the leaf area measurements. Statistical analysis of the results was performed using SPSS version 12.0.1 (SPSS, Chicago, Illinois, USA).

The number of Ki67 immunoreactive (-ir) cells in granule cell layer/subgranular zone (GCL/SGZ) was counted under 40x objective using a Nikon Microphot SA microscope. Cells were considered Ki67-ir if they were intensely stained and exhibited medium round or oval nuclear bodies. Ki67-ir cells were counted on half of every 6th section throughout the entire hippocampus. For representative Ki67-ir cells in the GCL/SGZ of the hippocampus, see Figure 1.

NSC-34-4#56 cells were treated with 2 μg/mL Dox for 3 days in 10% FBS/DMEM. Glass coverslips (Fisher 12-545-100) were sterilized by UV irradiation and were treated with 1.5 μg/mL Poly-D-Lysine. SMN depleted NSC-34 or control cells were plated onto coverslips at 3x10⁴ cells/well and reverse transfected with Lipofectamine 2000 and Rit and pEGFP-C1. After 3 days, cells were washed with PBS and fixed in 4% paraformaldehyde/PBS and permeabilized in blocking solution (PBS, 5% normal goat serum, 0.1% Triton X-100) for 30 min. Alpha-tubulin antibody was diluted 1:2500 in blocking solution and incubated with cells for 2 hrs followed by Alexa-Fluor 594 1:1000. Slides were mounted using Prolong gold with DAPI, then visualized on a Nikon Microphot-SA microscope. Neurite lengths were measured using QCapture Pro 6.0 software. All lengths were normalized to GFP control transfected cells.

NIKON-Microphot-SA microscope was used to capture images of DAB-stained embryos (40X). Imaging for all other smFISH and fluorescent immunostaining experiments was performed on a Nikon A1 inverted laser-scanning confocal microscope.
Images were assembled using ImageJ (NIH) and Adobe Photoshop and Illustrator software to crop regions of interest, adjust brightness and contrast, generate maximum-intensity projections, and separate or merge channels. To assess the mislocalization of the RNAs or protein in different genetic backgrounds compared to the control, we generated plot profiles using ImageJ. The posterior-most 75 μm of each embryo was plotted for comparison, and embryos from a single biological replicate are plotted in figures given that variation between fluorescence between replicates obscured the pole plasm distribution trends if embryos from all replicates were plotted together.

NIKON-Microphot-SA microscope was used to image and analyze DAB-stained embryos. Confocal imaging for all fluorescently labeled samples was performed on a Nikon A1 inverted laser-scanning confocal microscope. Unless noted in figure legend, all images were of single sections, not maximum intensity projections from Z stacks. To assess the spreading of the RNAs or protein in different mutant backgrounds compared to the control, we generated plot profiles using ImageJ. The posterior-most 75 µm of each embryo was plotted for comparison, and embryos from a single biological replicate are plotted in figures given that variation between fluorescence between replicates obscured the germ plasm distribution trends if embryos from all replicates were plotted together. Images were assembled using Fiji (ImageJ, NIH) and Adobe Photoshop software to crop regions of interest, adjust brightness and contrast, and separate or merge channels.