Except adult brain analysis, eGFP and tdTomato were detected by native fluorescence. Images are representative of at least two mice. For brain analysis, serial 60 μm thick coronal vibratome sections were generated and immunolabeled with anti-Tomato dsred as well as anti-Glial Fibrillary Acidic Protein (GFAP) antibodies, performed according to published protocols.25 ,36 (link) Immunolabeled sections were counterstained with Hoechst stain. Atlas images are from the Mouse Brain Library (http://www.mbl.org/mbl_main/atlas.html ).37 Fluorescent microscopy of slides was performed using either Zeiss LSM-700 Confocal Microscope Carl Zeiss AG, Oberkochen, Germany) (single plane) and or on a Zeiss Axio Imager M2 with respective ZEN software. Differential interference contrast microscopy of slides was performed using Zeiss Axio Imager M2. Whole-mount brightfield images were performed using an Olympus SZX12 Stereozoom microscope (Olympus Corporation, Tokyo, Japan) or Olympus MVX10 microscope. Whole mount fluorescent images were performed using an Olympus MVX10 microscope excited using an X-Cite Turbo XT600-T fluorescence light source (Excelitas Technologies Corp, Waltham, Massachusetts) at 475 and 575 nm wavelengths. Images were processed using either Canvas X (Canvas GFX), ImageJ, or Photoshop (Adobe Inc., San Jose, California).
Szx12 stereozoom microscope
Manufactured by Olympus
Sourced in Japan
The SZX12 stereozoom microscope is a versatile imaging instrument designed for a wide range of applications. It features a zoom ratio of 12.5:1, providing a magnification range of 0.8x to 10x. The microscope utilizes an optical system that delivers high-quality, true-to-life images with excellent clarity and resolution. The SZX12 is suitable for use in various laboratory settings, offering a reliable and efficient tool for detailed observation and analysis.
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7 protocols using szx12 stereozoom microscope
1
Multimodal Imaging of Transgenic Mice
2
Oral Bacterial Infection in Adult Flies
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Briefly, prior to infection, adult flies of appropriate genotype were starved for 2 hr, then transferred into vials containing filter paper hydrated with 5% sucrose solution mixed with concentrated Ampicillin resistant E. coli (A600 = 1; concentrated to contain ~10 CFU/ml) or Gram positive bacteria (B. subtilis) A600 = 5–10) on cornmeal food. Following incubation at 25 °C for 24 hr, flies were processed for RNA extraction or examined for reporter-GFP expression. For survival assay, flies were challenged with bacteria by oral feeding. Adult flies were starved for 48 hours and then transferred to fresh corn-meal food vials containing fresh filter paper disks inoculated with bacterial cultures. The percentage of survivors was then calculated for each experiment and plotted as a survival curve. (N = 100) for each genotype. Reporter-GFP expressing flies were imaged on an SZX12 stereozoom microscope (Olympus) and processed uniformly for brightness/contrast using Adobe Photoshop CS3.
3
Visualizing Crystal Cells in Drosophila Larvae
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Crystal cells are characterized by crystalline inclusions that contain the zymogen ProPO and can be visualized due to specific blackening upon heating larvae at 60 °C for 10 min64 (link). Third instar wandering stage larvae were heat treated to visualize crystal cells and imaged using SZX12 stereozoom microscope (Olympus). Melanised crystal cell were counted from three posterior abdominal segments of at least 20 larvae per genotype. Error bars represent the standard deviation. P-values were calculated using one way ANOVA.
4
Measuring Phenoloxidase Activity in Insect Hemolymph
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For the measurement of PO activity by dot blots, 5 µl hemolymph was applied to a filter paper pre-soaked with 20 mM L-DOPA (L-3, 4-dihydroxyphenylalanine- Cat. No. D9628, SIGMA) in phosphate buffer pH 6.6, incubated for 20 minutes at 37 °C and heated in a microwave till the paper was dried completely65 (link). The melanised black hemolymph spots correlate with PO activity in hemolymph and were imaged under an Olympus SZX12 stereozoom microscope.
For photometric measurement of PO activity, 10 µl hemolymph from each strain was pooled on ice by quickly bleeding 3–5 larvae and withdrawing 6 µl hemolymph. Aliquots of mixed hemolymph were activated at 25 °C for 10 minutes, then 40 µl L-DOPA was added and optical density (OD) measured from 0 to 30 minutes at 490 nm with a VmaxTM Kinetic Microplate Reader (BIO-RAD). Activation of PO was measured as the relative change in absorbance (A490). Experiments were repeated at least three times with biological and technical replicates.
For photometric measurement of PO activity, 10 µl hemolymph from each strain was pooled on ice by quickly bleeding 3–5 larvae and withdrawing 6 µl hemolymph. Aliquots of mixed hemolymph were activated at 25 °C for 10 minutes, then 40 µl L-DOPA was added and optical density (OD) measured from 0 to 30 minutes at 490 nm with a VmaxTM Kinetic Microplate Reader (BIO-RAD). Activation of PO was measured as the relative change in absorbance (A490). Experiments were repeated at least three times with biological and technical replicates.
5
Staining Phytoplasma-Infected Tomato Leaves
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At 60 dpi, the mature leaves of tomato plants (cv. MM) infected with PPT phytoplasma and mock inoculated controls were collected during the day and at the end of darkness period. The collected leaves were cleared in 70% ethanol at 80 °C for 15 min, and stained with Lugol’s solution (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature, and then rinsed with water for 15 min. The stained leaves were observed and imaged with an Olympus SZX12 stereo zoom microscope (Olympus, Tokyo, Japan).
6
GUS Staining of Transgenic Tomato Lines
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GUS staining was carried out according to the protocol previously described with minor modifications [56 (link)]. Leaf axils and inflorescences from infected transgenic DR5::GUS reporter and ARR5::GUS tomato lines and mock control plants were collected, respectively. The samples were incubated in a GUS staining buffer (50 mM phosphate buffered saline [PBS], pH 7.2, 0.05% Triton X-100, 2 mM potassium ferrocyanide, 2 mM X-Gluc) for 4 h at 37 °C in the dark. The stained tissues were then bleached with a graded ethanol series at room temperature until chlorophyll was completely removed. The samples were then photographed with an Olympus SZX12 stereo zoom microscope (Olympus, Tokyo, Japan).
7
Quantifying Islet Graft Antibody Labeling
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