Axioplan2 epifluorescent microscope

Multinucleated giant cells (MGCs), generated by stimulated RAW 264.7 cells on coverslips in 24-well tissue culture dishes, were stained for tartarate-resistant acid phosphatase using the Acid Phosphatase (TRAP), Leukocyte Kit (Sigma, Oakville, ON). Briefly, cells were fixed for one minute with the fixative solution at room temperature, followed by staining with the staining solution for 1 hour at 37°C. Following water washes, coverslips were air dried and analyzed microscopically using a Zeiss Axioplan 2 epifluorescent microscope with a black and white AxioCam HRm and colour AxioCam HRc. MGCs generated by RAW 264.7 cells on Osteo Assay Surfaces were stained stained similarly with the exception that cells were fixed with 4% PFA instead of the suggested fixative solution.
The fusion index was calculated by dividing the number of nuclei in MGCs by the total number of nuclei in the field of view. The index was averaged over four independent experiments, with 40–50 MGCs analyzed per experiment.

Cells were seeded on 22x22mm glass coverslips in serum. The cells were fixed in either 4% paraformaldehyde (vol/vol) at room temperature or methanol at -20°C for 10min depending on the required primary antibody to be used, after which the cells were rinsed and permeabilized in 0.5% TritonX-100 in PBS for 10min. Cells were then blocked with 10% FBS for 10min. Primary antibody diluted in 0.5% TritonX-100 in PBS was added for 2h at room temperature and secondary antibody was diluted similarly and incubated for 1h as well. Coverslips were mounted with fluorescent mounting medium (Thermo Scientific) and visualized using a Roper Scientific CoolSnap HD digital camera adapted to Zeiss Axioplan2 epifluorescent microscope at 63X magnification. Image analyses were performed using Olympus FV10-ASW 1.6 viewer, Image-Pro Plus (Media Cybernetics, Inc, MD) or ImageJ (NIH, USA) software.

Tibialis anterior (TA) muscle sections were prepared for staining essentially as previously described^{6 (link),48 (link)}. For OSMR staining, whole TA muscles were first fixed with 0.5% paraformaldehyde/PBS for 2 h at 4 C, followed by incubation in 20% sucrose/PBS overnight at 4 C. 10 μM sections were cut for all stainings. Antibodies were: anti-laminin (Millipore, catalog #05⁻206, 1:250), anti-GFP (Invitrogen, catalog #A11122, 1:200), anti-OSMR (R&D, catalog #AF665, 1:200), anti-Pax7 (Santa Cruz Biotechnology, catalog #sc81648, 1:50; or Developmental Studies Hybridoma Bank, 2 μg/mL final concentration), AlexaFluor 594–conjugated donkey anti-rat IgG1 and AlexaFluor 488–conjugated donkey anti-rabbit (Jackson ImmunoResearch, catalog # 712-585-150 and 711-545-152 respectively, 1:200 each). Nuclei were counterstained with either DAPI (Invitrogen) or TO-PRO-3 (Invitrogen). Images were acquired with an AxioPlan2 epifluorescent microscope (Carl Zeiss) with ORCA-ER digital camera (Hamamatsu Photonics).

Slides containing rabies virus tracings were imaged using a 5x/0.15 NA objective on an Axioplan2 epifluorescent microscope (Zeiss, Jena, Germany) equipped with a Ludl controllable stage (Visitron Systems, Puchheim, Germany), a CoolSnapHQ² CCD camera (Teledyne Photometrics, AZ), and orchestrated by µManager 2.0 beta software (Edelstein et al., 2014 (link)). Excitation was provided by an X-cite halogen lamp (Excelitas Technologies, MA) with 350/50x (DAPI) and 470/40x (eGFP) filter cubes.
Axonal AAV tracings were imaged on an SP5 or SP8 laser scanning confocal microscope (Leica, LAS AF and LAS X 3.5.0.18371, respectively) using a 10x/0.40 NA objective, and a 1 Airy disc pinhole. 405 nm and 488 nm laser lines were used to image DAPI and eYFP channels. Single optical z-section images of 10 µm thickness from the middle (z-axis) of the section were acquired. For each brain, we determined the densest efferents outside the insular cortex, and adjusted the acquisition settings to obtain a nearly saturated signal.
Starter volumes for RV tracings were determined by imaging sections covering the injection site with an SP5 microscope using the 10x objective. 10 z-stacks of 7 μm step-size through each section were acquired. For AAV starter cells, sections covering the injection site were imaged as a single plane on the epifluorescent microscope with a 5x objective.

Wandering 3^rd instar larvae were dissected in PBS, fixed in 4% formaldehyde, washed in PBS, and mounted in vectashield mounting media with DAPI (Vector Labs). Images were collected on a Zeiss LSM 510 confocal microscope. GFP was excited with a 488 nm argon laser and a HFT 488 primary dichroic. GFP emission was filtered with NFT 490 and BP 500–550 IR filters before collection. DAPI was excited with a chameleon two photon laser (Coherent) at 720 nm and a HFT KP 650 primary dichroic. DAPI emission was filtered with a 480–520 IR filter before collection.
Embryos were collected on grape juice plates, dechorionated, fixed, and mounted in vectashield with DAPI. Images were collected on a Zeiss Axioplan2 epifluorescent microscope. Images were deconvoluted with MetaMorph (Molecular Devices).
Wings were removed from adult flies in ethanol, mounted in euparal, and incubated overnight at 37°C. Wings, larvae, and pupal cases were imaged on a Leica MSV269 stereoscope.

Fluorescent images were acquired on an LSM 710 microscope (Zeiss, Oberkochen, Germany). Chromogenic ISH images were obtained on an Axioplan2 epifluorescent microscope (Zeiss, Oberkochen, Germany). Whole-clutch bright-field images were captured using a SteREO Discovery.V12 (Zeiss, Oberkochen, Germany) and AxioCam MRc5 camera (Zeiss, Oberkochen, Germany). Images were processed using ImageJ software (National Institutes of Health) and Imaris (Bitplane AG, Zurich, Switzerland).
Signal intensity measurements to assess RNAscope probe penetration were performed on confocal images after subtracting background levels using the rolling ball algorithm. The measurements were performed at four different selected expression sites in the vessels. Measurements were repeated for three independent embryos. Signal-to-noise ratio was determined employing the ROI manager of ImageJ, dividing the average pixel intensity of an area expressing egfp under the control of the fli1a promoter with that in a region where egfp is not expressed. This procedure was repeated six times in different confocal planes.