The genetic screen which led to the isolation of the inp2-1 mutant was recently described21 (link). In brief, M2 plants (~10,000) from eight pools of ethylmethane sulfonate (EMS)-treated lines of Landsberg erecta background were screened for the presence of morphological abnormalities in their pollen (e.g. in size, shape, light reflection, ease of pollen release from anthers) identifiable with standard dissecting stereomicroscopes (Zeiss Stemi-2000C and Nikon SMZ745) at 75-80X magnification. Particular attention was paid to changes in pollen shape, known to be associated with aperture defects9 (link),13 (link). For primary screening, dry pollen did not undergo any treatment. At this level of magnification, pollen of the inp2-1 mutant looked rounder than the wild-type pollen. Pollen was then stained with auramine O as described14 (link), and aperture defects were observed with confocal microscopy. inp2-1 was then backcrossed with Ler once. To test for complementation, inp2-1 mutant was crossed with inp1-1, and the pollen of their F1 progeny was observed with dissecting and confocal microscopes.
Stemi 2000c
Manufactured by Nikon
The Stemi-2000C is a stereo microscope designed for observing and examining samples. It features a trinocular viewing head, a zoom range of 6.3x to 50x, and a built-in LED illumination system. The Stemi-2000C is suitable for a variety of applications, including material inspection, biological sample analysis, and educational purposes.
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7 protocols using stemi 2000c
1
Genetic Screen for Pollen Mutants
2
Genetic Screen for Pollen Mutants
3
Adipose Tissue Analysis in Peripheral Nerves
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Screening for Arabidopsis Pollen Mutants
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Wild-type (Landsberg erecta) and mutant Arabidopsis plants were grown at 20 − 22° C with the 16-hour light: 8-hour dark cycle in growth chambers or in a greenhouse at the Biotechnology facility at OSU. The genetic screen that led to the isolation of the mcr and dnt mutants was performed similar to the previous screen [42 (link)]. In brief, M2 plants from eight pools of EMS-treated lines of Landsberg erecta background (~10,000 plants) were screened for the presence of morphological abnormalities in their pollen (e.g. in size, shape, light reflection, ease of pollen release from anthers) that were identifiable with standard dissecting stereomicroscopes (Zeiss Stemi-2000C and Nikon SMZ745) at 75-80X magnification. For primary screening, pollen did not undergo any treatment. Particular attention was paid to changes in pollen shape, known to be associated with aperture defects [12 (link), 42 (link)]. Pollen of the candidates isolated in the primary screen was stained with auramine O as described [13 (link)] and observed for exine and aperture defects with confocal microscopy. Confirmed mutants were then backcrossed at least once. mcr and dnt mutants were crossed with the previously described DMC1pr::INP1-YFP line [14 (link)] and with osd1-2/+ plants that were identified as described [34 (link)].
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Optokinetic Response in Zebrafish
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Adult zebrafish were lightly anaesthetized in 0.2 mg/ml tricaine and placed in a custom-made foam holder supported by dissection pins in a 55 mm petri dish. The dish was filled with tank water and the fish were allowed to regain consciousness, then placed into a custom-made optokinetic device consisting of a 12 cm acrylic drum, rpm adjustable rotating motor with laser tachometer and stereo microscope (Zeiss Stemi-2000C) c-mounted with a digital SLR camera (Nikon D5100). Each zebrafish was assessed with varying grating lengths at a consistent rpm speed (12 rpm) until the stripes could no longer be tracked by the zebrafish, as previously described (52 (link)).
6
Sexual Size Dimorphism in Spiders
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For laboratory reared spiders, body mass and size are correlated [66] . We assessed sexual size dimorphism based on mass and size for spiders reared under the same conditions for a sample of lab-reared spiders. All spiders (N for females, males: L. geometricus = 45, 68; L. hasselti = 42, 72; L. mirabilis = 46, 71; L. mactans = 23, 22; and L. hesperus = 42, 78) were weighed using an analytical balance (Ohaus electronic balance, accurate to 0.01 mg). For the estimation of body size, spiders (N for females, males: L. geometricus = 20, 22; L. hasselti = 21, 20; L. mirabilis = 20, 21; L. mactans = 17, 16; and L. hesperus = 22, 26) were photographed using a dissecting microscope (Zeiss Stemi 2000-C) and a digital camera (Nikon DXM 1200). Photos were taken of the female's and male's patella-tibia length in both left and right front legs, which provides an accurate representation of body size in spiders [47] . The photos were later analyzed and measured using Image J. Sexual dimorphism was calculated as female mass/ male mass, and female size/male size independently. We also report the coefficient of variation for mass and size (CV = standard deviation/mean). This measure of variation can be meaningfully compared across groups with different mean sizes.
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Optokinetic Response Assay in Zebrafish
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WT and obetd15 zebrafish were lightly anaesthetized in 0.2 mg/ml tricaine and placed in a custom-made foam holder supported by dissection pins in a 55 mm petri dish. The dish was filled with tank water and the fish were allowed to regain consciousness, then placed into a custom-made optokinetic device consisting of a 12 cm acrylic drum, rpm adjustable rotating motor with laser tachometer and stereo microscope (Zeiss Stemi-2000C) c-mounted with a digital SLR camera (Nikon D5100). Each zebrafish was assessed with varying grating lengths (from 0.4 cm to 0.04 cm in 0.04 cm increments) at a consistent rpm speed (12 rpm) until the stripes could no longer be tracked by the zebrafish, following published protocols70 . Visual acuity was calculated as cycles per degree (cpd) using the following equation: where a is the distance from the center of the lens to the grating, and h is the length of one cycle of the smallest grating at which an optokinetic response was observed. As a positive control, three WT zebrafish at 6 mpf received an intravitreal injection of 0.1 ml of 10 µm ouabain to induce a chemical retinal degeneration, and were assessed at day 3 post-injection, a stage at which the retina is known to be ablated71 (link).
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