GUS activities in RSS1pro:GUS, DR5:GUS and rss1xDR5:GUS lines under various growth and treatment conditions were determined using a standard GUS histochemical staining procedure as described43 (link). Seedlings after treatment were subsequently incubated at 37 °C in a GUS staining solution [0.1 M sodium phosphate buffer, pH 7, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 50 mM EDTA, and 1 mg mL21 5-bromo-4-chloro-3-indolyl-b-glucuronic acid] for 30 min to 40 min. The seedlings were then kept in 70% (v/v) ethanol for the removal of chlorophyll. The staining in the seedlings was then observed under Microscopy was done on Zeiss Axio Imager2 microscope using differential interference contrast (DIC) optics (Carl Zeiss, Germany) or on Nikon SMZ1500 Stereo-Zoom microscope, and photographs were taken with a Nikon Coolpix digital camera connected to a Nikon SMZ1500 Stereo-Zoom microscope. The experiment was repeated thrice, with each replicate having at least 10 seedlings, yielding similar results.
Smz1500 stereo zoom microscope
Manufactured by Nikon
Sourced in Germany
The SMZ1500 Stereo-Zoom microscope is a laboratory equipment designed for observation and examination of samples. It provides variable magnification with a zoom ratio of 15:1, allowing for detailed inspection of specimens. The SMZ1500 features a sturdy, ergonomic design to support research and analysis activities.
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6 protocols using smz1500 stereo zoom microscope
1
Visualizing GUS Expression Patterns
2
Effects of dsRNA on Insect Fertility
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Thirty female adults in each treatment group were used to assess effects on survival rates. The experiment was repeated three times.
For tissue observations, 10 female adults at different d.p.i. were used for dissection. The females were dissected in phosphate buffer solution under a Nikon SMZ1500 stereozoom microscope (Nikon, Tokyo, Japan) and photographed with the NIS Elements software (Nikon, Tokyo, Japan). The number of ovarioles at different vitellogenic stages was counted.
For fertility analysis, each treated female adult was place with two untreated males in a 50 mL centrifuge tube containing fresh rice seedlings that were changed every two days within the 10 d.p.i., and the replaced rice seedlings were maintained independently in new 50 mL tubes. The number of nymphs newly hatched on the seedlings was counted every day until no nymphs hatched, and finally, the seedlings were dissected under the Nikon SMZ1500 stereozoom microscope to observe and count the unhatched eggs. Fifteen female adults were assessed from the two dsRNA-treated groups, and three repetitions were performed.
For tissue observations, 10 female adults at different d.p.i. were used for dissection. The females were dissected in phosphate buffer solution under a Nikon SMZ1500 stereozoom microscope (Nikon, Tokyo, Japan) and photographed with the NIS Elements software (Nikon, Tokyo, Japan). The number of ovarioles at different vitellogenic stages was counted.
For fertility analysis, each treated female adult was place with two untreated males in a 50 mL centrifuge tube containing fresh rice seedlings that were changed every two days within the 10 d.p.i., and the replaced rice seedlings were maintained independently in new 50 mL tubes. The number of nymphs newly hatched on the seedlings was counted every day until no nymphs hatched, and finally, the seedlings were dissected under the Nikon SMZ1500 stereozoom microscope to observe and count the unhatched eggs. Fifteen female adults were assessed from the two dsRNA-treated groups, and three repetitions were performed.
3
Quantifying Auxin-Responsive GUS Activity
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GUS reporter activity was determined using a standard GUS histochemical staining procedure. Briefly, 5-d-old light-grown DR5::GUS seedlings were transferred to 1/2 MS growth medium supplemented without or with Glc (0% and 3% [w/v]) and solidified with 0.8% (w/v) agar for 4 d. Seedlings after treatment were subsequently incubated at 37°C in a GUS staining solution [0.1 m sodium phosphate buffer, pH 7, 0.5 mm K 3 Fe(CN) 6 , 0.5 mm K 4 Fe(CN) 6 , 50 mm EDTA, and 1 mg mL -1 5-bromo-4-chloro-3-indolyl-β-glucuronic acid] for 6 h. The seedlings were then kept in 70% (v/v) ethanol for the removal of chlorophyll. Different stages of LR development were then observed and scored using a Nikon SMZ1500 Stereo-Zoom microscope, and photographs were taken with a Nikon Coolpix digital camera connected to a Nikon SMZ1500 Stereo-Zoom microscope. The experiment was repeated twice, with each replicate having at least 10 seedlings, yielding similar results.
4
Morphological Observation of Ant Specimens
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The morphological observation was conducted on a Nikon SMZ 1500 stereo zoom microscope. For digital images, MP evolution digital camera was used on the same microscope with Auto-Montage (Syncroscopy, Division of Synoptics, Ltd.) software. Later, images were cleaned as required with Adobe Photoshop CS5.
Abbreviations of the specimen depositories are:
BMNH The Natural History Museum, London, England, U.K.
MRSN Museo Regionale di Scienze Naturali, Torino, Italy.
NHMB Naturhistorisches Museum Basel, Switzerland.
PUPAC Punjabi University Patiala, Ant Collection, Patiala, India.
ZSIK Zoological Survey of India, Kolkata, India.
Abbreviations of the specimen depositories are:
5
Immunolocalization of Auxin in Arabidopsis
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For free IAA accumulation estimation, 7 days old seedlings of WT (Col-0), rss1 and RSS1OE were kept in histochoice clearing agent (sigma) for 20 min followed by washing twice with 100% ethanol for 5 min each. Tissues/samples were rehydrated by passing through gradients of ethanol (95%, 80%, 60%, 30% and sterile water) for 5 minutes each and followed by PBS wash; (2 × 10 minutes). Whole seedling IAA immunolocalization was performed using monoclonal anti-auxin antibody (A0855, sigma) as described previously74 . Calorimetric detection was performed using NBT/BCIP based detection solution (Roche Diagnostics, India), as per company’s manual. Once visible signal was observed, seedlings were washed with PBS (2 × 10 minutes) followed by dehydration increasing gradients of ethanol (5 min each) and kept in histochoice clearing agent for 10 min. Seedlings were mounted on glass slides with 10% glycerol and imaged using a Nikon Coolpix digital camera connected to a Nikon SMZ1500 Stereo-Zoom microscope.
6
Pollen Quantification and Oxidative Stress
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Pollen grains were diluted to 10 μg/μl in PBS and boli were crushed in 600 μl for the Amplex Red assay above. For pollen from midgut contents, an aliquot of 50 μl was stained with Safranin O for pollen counting (Jones 2012) (link). Pollen counts for a known area of the slide were made using a Nikon SMZ1500 stereo zoom microscope and multiplied by the whole slide area. The total number of pollen grains was calculated using total volume of the sample and total area of the slide.
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