Stereo lumar v12

To analyze lymph transport by mesenteric lymphatic vessels, mice received intragastrically 0.1 mg of BODIPY FL C₁₆ (4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-hexadecanoic acid, Molecular Probes, Inc.) in heavy cream. Mice were sacrificed 2 h later and transport of fluorescent lipid visualized by Zeiss Stereo Lumar v.12 fluorescence stereomicroscopy.

For GUS staining, various tissues including four-leaf-stage root; tillering stage stem and leaf; heading stage flag leaf; a hull before flowering; flower; grain-filling-stage developing seeds (3–5, 10–12, and 15–17 days after flowering); and germinating seeds at 24 h, 36 h, 2 days, 3 days, 4 days, 5 days, and 6 days from the pOsGrxC2.2:GUS transgenic plants were incubated in a solution containing 50 mM sodium phosphate (NaPO₄) buffer (pH 7.0), 5 mM potassium ferricyanide (K₃Fe(CN)₆), 5 mM potassium ferrocyanide (K₄Fe(CN)₆), 0.1% Triton X-100, 20% (v/v) methanol (MeOH), and 1 mM X-Gluc and stained at 37°C for 6 h and destained twice with 75% ethanol. They were then observed under a stereoscopic microscope SteREO Lumar V12 (Carl Zeiss, Heidenheim, Germany).

GFP and alizarin red images were captured using a Zeiss SteReo Lumar.V12 equipped with a Zeiss AxioCam HRc digital camera and Zeiss AxioVision Rel. 4.8 software.

All of the P. vitticeps, Anolis carolinensis, Boaedon fuliginosus, and Pantherophis guttatus eggs were obtained from our animal facility at the University of Helsinki. Eggs from Lampropeltis getula, Python sebae, and Furcifer pardalis were obtained from reptile breeders or Tropicario Helsinki. Fertilized eggs were incubated on a moistened vermiculite substrate at 29.5°C (or 26°C for F. pardalis) until opened. P. vitticeps embryos were collected from different females and over three breeding seasons to obtain consistent and accurate staging. Early and/or small embryos were imaged using a stereomicroscope (Zeiss SteREO Lumar V12, Objective: ApoLumar S 1.2 FWD 47 mm, Camera: Axiocam ICc 1) to create z-stacks, and stacks were combined and converted into TIFF files using Zeiss Zen 2.3 (Blue edition). Larger embryos were imaged using a Nikon D3200 digital camera and incident light. Photographs of other embryonic species were sampled from published literature.

Following in situ hybridization, larvae at stages 37–44 were dissected at the level of the jaw joint to enable whole mount visualization of teeth that had developed on the roof and floor of the mouth. Additionally, mouth roof epithelia of embryos hybridized with the Shh probe were excised and photographed as flat-mounts to prevent masking of the tooth-specific signal from the signal in the overlying neural tube. Whole mounts processed through in situ hybridization, skeletal stainings, and transplantation of GFP-labeled oral ectoderm were photographed as Z-stacks using motorized dissection microscopes (Olympus SZX12, Zeiss SteREO Lumar.V12). The final deep-focus images were acquired by merging the Z-stacks using maximum projection function. Larvae processed through transplantation of GFP-labeled oral ectoderm were further sectioned using the CM3050 cryomicrotome (Leica) and individual tooth germs were visualized using an anti-calbindin antibody (Sigma) as previously described (Barlow and Northcutt, 1997 (link); Soukup et al., 2008 (link)). Z-stacks of sections were taken on the Olympus Cell^R IX81 microscope and merged as maximum projection images.