Eclipse ni e upright microscope

Because the first exon of Gdf15 is predicted to be expressed in Gdf15 knock-out first mice, we requested a custom RNAscope probe design that targeted the second exon of Gdf15 (Mm-Gdf15-O2; targeting c.764-1534 of NM_011819.3). This custom probe was used in conjunction with the RNAscope 2.5 High Definition-BROWN assay to stain formalin-fixed paraffin embedded liver sections according to the manufacture’s recommendations. Slides were counterstained with hematoxylin QS (Vector laboratories). For analysis of Rosa^mTmG mice, livers were fixed in 4% PFA for two hours prior to cryoprotection in 30% sucrose in PBS overnight. The following morning, livers were embedded in OCT and sectioned. Sections were washed in PBS, stained with DAPI, and imaged immediately. All images were captured with a Nikon Eclipse Ni-E upright microscope equipped with both ORCA-FLASH 4.0 and DS-Fi3 cameras for widefield and brightfield imaging, respectively.

Wide-field images were acquired on a Nikon Eclipse Ni-E upright microscope equipped with a CFI Plan Apochromat Lambda 20× 0.75 NA objective lens (Nikon), Nikon motorized XY stage, and Andor Zyla 4.2 Plus scientific CMOS camera, driven by NIS-Elements. Alexa 647–labeled bovine tubulin, EB1-GFP (110 nM final), Tau-mCherry (20 nM final; Mooney et al., 2017 (link)), and/or Alexa 568–labeled 10 kDa dextran were supplemented to the reactions. Dark current subtraction and flat field correction were applied to both channels. A custom script written in MATLAB was used to quantify the fluorescent intensity as a function of radial distance from the MTOC (Pelletier et al., 2020 ). Alexa 568–labeled dextran showed a flat intensity profile with respect to the aster as expected for a molecule that shows no spatial variation (Supplemental Figure 4). Spot detection of EB1 comets was performed with TrackMate as described above.

Cell proliferation in E16.5 lung cryosections was characterized by BrdU and phospho histone H3 immunofluorescence and quantified in Nikon Elements General Analysis 4.50. For BrdU labeling, E16.5 pregnant mice were injected intraperitoneally with BrdU at 50 mg/kg of body weight and the animals were sacrificed 2 hours after the injection. For quantification of immunofluorescence staining, tile scans of the whole lung were taken on a Nikon A1Rsi inverted laser confocal microscope; approximately 10, 40× random regions were selected. In each image, an average of 1,000 cells was counted for analysis. A total of 10,000–15,000 cells were counted per mouse. A 2-tailed Student’s t-test was used to determine statistical differences between genotypes. Cell death in E16.5 paraffin sections were characterized by TUNEL assay and Caspase-3 immunohistochemistry and quantified in Nikon Elements General Analysis RGB 4.50. Widefield tile scans were taken of the whole lung on a Nikon Eclipse Ni-E upright microscope; 10–15, 20X random alveolar regions were selected. In each image, an average of 2,000 cells was counted for analysis. A total of 20,000–30,000 cells were counted per mouse. A 2-tailed Student’s t-test was used to determine statistical differences between genotypes.