The nematodes were treated as described above and were maintained on NGM/E. coli OP50 plates until they reached larval stage L4. To evaluate progeny size, one nematode from each treated group was transferred to a new NGM plate seeded with E. coli OP50 with or without 100 mM glucose supplementation along with Madhugrit (3, 10, and 30 μg/ml) or Metformin (2 mM). The total number of progenies were counted by ZEISS Stemi 305 stereo microscope (Carl Zeiss, Germany). Data were presented as mean ± SEM (n=5).
Stemi 305 stereomicroscope
Manufactured by Zeiss
Sourced in Germany
The Stemi 305 is a stereomicroscope from Zeiss. It provides stereoscopic, high-resolution imaging for observation and documentation of specimens.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam erc 5s microscope camera, by Zeiss (2 mentions)
Zc0123, by Vector Laboratories (1 mentions)
C5042, by Merck Group (1 mentions)
Methylene blue, by Merck Group (1 mentions)
Coomassie brilliant blue, by Merck Group (1 mentions)
Powershot g7 x mark 2, by Canon (1 mentions)
Axiocam 105 colour camera, by Zeiss (1 mentions)
Congo red, by Merck Group (1 mentions)
Calcofluor white, by Merck Group (1 mentions)
Genteal eye gel, by Alcon (1 mentions)
Tropicamide, by Alcon (1 mentions)
Alcaine, by Alcon (1 mentions)
Phenylephrine hydrochloride, by Alcon (1 mentions)
Agar, by Helicon (1 mentions)
11 protocols using stemi 305 stereomicroscope
1
Evaluating Nematode Progeny Size
2
Trap Formation in Arthrobotrys oligospora
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A. oligospora only develops traps in low nutrient environments in the presence of nematodes (4). Consequently, A. oligospora strains were first cultured for 2 days in 3.5 cm diameter LNM-containing Petri-dishes. Then, 30 adult living C. elegans nematodes were placed on the Petri-dishes for 6 hour and subsequently washed out from the plates with M9 buffer (22 mM KH2PO4, 42 mM Na2 HPO4, 86 mM NaCl). After an additional 18 hours of growth, A. oligospora strains were imaged using a Zeiss Stemi 305 Stereo Microscope (Zeiss, Göttingen, Germany) and a Zeiss Axiocam ERc 5s Microscope Camera (Zeiss, Göttingen, Germany). Three 40X images of the mycelium (size 2.5 x 2.1 mm) per plate were captured at random for 6 plates per strain, yielding a total of 18 images per strain.
3
Fungal Conidia Isolation and Imaging
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Spore suspension of fungal strains was inoculated on 5 cm PDA (YPD for N. crassa) plates and incubated for 5 days at 25°C in the dark. Next, 2 ml of ddH2O was added to each plate and conidia were carefully scratched off from the surface of the mycelium. The ddH2O from the plate was first filtered through two layers of non-woven cloth (to exclude extraneous hyphae) and then transferred to a 2 ml centrifuge tube for centrifugation at 13,000 x g for 1 minute. For each tube, 1 ml of supernatant was extracted and 10 x 5 μl droplets of the remaining solution were placed on an LNM Petri-dish. Each droplet was imaged using a Zeiss Stemi 305 Stereo Microscope (Zeiss, Göttingen, Germany) and a Zeiss Axiocam ERc 5s Microscope Camera (Zeiss, Göttingen, Germany) at a resolution of 80X and 16X, for the conidiation study of fungal species and A. oligospora strains, respectively. The images comprised 2290×1920 pixels, representing an area of about 1.3 x 1 mm for the 80X images and 6.3 x 5.3 mm for the 16X images.
4
Experimental Lung Metastasis Assay
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PAR, ICD–CSCs, Kdm1bKD and Kdm1bOVER MCA205 cells (2 × 105) were injected into the tail vein of C57Bl/6J mice. In some experiments, mice were treated with 200 µg per mouse anti-CD4 and anti-CD8 Abs in D-PBS, at day-1 and then every 4 days for 15 days. Then, lungs were explanted and macrometastases counted. For CD4–CD8 in vivo depletion, at the end of the experiments, spleens were recovered and analyzed by cytofluorometry. Images of lung metastases were captured with a ZEISS STEMI 305 Stereo microscope (Carl Zeiss). GraphPad Prism was used for data analysis.
5
Cresyl Violet Staining of Brain Tissue
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Tissue slices (30 µm thickness) were stained with 0.1% fast cresyl violet acetate (C5042, Sigma-Aldrich, St. Louis, MO, USA). Slices were cleared under xylene for 20 min after dry in the air, then rehydrated for 2 min using 100% ethanol to 50% ethanol. After staining in cresyl violet for 2 min and a progressive dehydration protocol procedure. Slices were cleared in xylene for 5 min before covering with mounting medium (ZC0123, Vector Laboratories, Burlingame, CA, USA). Images were taken with ZEISS Stemi 305 Stereo Microscope (Carl Zeiss AG, Oberkochen, Germany).
6
Clonogenic Survival Assay for Radiotherapy Resistance
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Resistance to therapy was determined by clonogenic survival assays as described [7 (link)]. In brief, cells were seeded into 6-well plates and incubated for 4 h in order to adhere. Cells were irradiated at the indicated doses in single-shot or in intervals of 24 h (fractionated mode), and colony formation was allowed for up to 12 d. In case of TMZ treatment, cells were incubated with TMZ at the indicated doses for 24 h, medium was changed, and colony formation was performed in TMZ-free medium. For the combination treatment, cells were exposed to 5 µM TMZ for 24 h, irradiated at the indicated doses, and incubated in TMZ-free medium for colony formation. Colonies were fixed with 80% ethanol, stained with 0.8% methylene blue (both from Merck Millipore, Darmstadt, Germany), and counted with a Stemi 305 stereomicroscope (Carl Zeiss, Oberkochen, Germany) as described [7 (link), 14 (link)]. Percentages of colony forming cells were calculated and normalized to the respective plating efficiencies at approximately matched colony numbers. Resistance scores were extracted by principal component analysis (PCA) as scores of the first principal component (PC1) [8 (link)].
7
Measuring C. elegans Reproductive Output
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Bacteria were grown as described in “Bacterial Culture Conditions”. Synchronized L1s were plated on NGM containing bacteria with indicated concentrations of sodium butyrate and incubated for 48 h. After 48 h, worms were replated every 24 h until they were 96 h old. The number of adult worms per plate was accounted for after each transfer. To count the sum of the eggs and L1 larvae laid by gravid adults between 48–72 and 72–96 h of age, entire plates were washed with M9, pipetting up and down meticulously to ensure removal of eggs and larvae. Plates were verified void of eggs and larvae by confirming their removal under the Zeiss Stemi 305 stereo microscope. Eggs and larvae were washed twice with 15 mL M9 and were resuspended in 2 mL M9. Ten or 100 μL samples of M9 were taken from the falcon tube and eggs + L1-stage worms were counted in replicates of six per run. The total number of offspring was calculated by factoring in the dilution and was expressed as the total number of progenies per worm.
8
Visualization of Salmonella Enteritidis Colony Morphology
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For analysis of colony morphology, 1 µl of overnight cultures of the S. Enteritidis strains was used to spot onto LB agar plates without salt, containing 40 µg ml−1 of Congo red (Sigma) and 20 µg ml−1 of Coomassie brilliant blue (Sigma) (CR agar plates) or 200 µg ml−1 of calcofluor white (Sigma) (CFW agar plates). The resulting colonies were grown at 26 °C for 96 h and visualized using a Stemi 305 stereomicroscope (Zeiss) equipped with an Axiocam 105 colour camera (Zeiss) or photographed using a digital camera (PowerShot G7X Mark II; Canon).
9
Intravitreal Injection in C57BL/6J Mice
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Ten- to twelve-week-old C57BL/6J mice were anesthetized with a ketamine/xylazine cocktail (120 mg/kg, 16 mg/kg, respectively) followed by pupil dilation using tropicamide (1% [w/v]) and phenylephrine hydrochloride (2.5% [w/v]), both from Alcon, Fort Worth, Texas. Alcaine (0.5% [w/v], Alcon) was used to locally anesthetize the eye followed by application of GenTeal eye gel (severe dry eye formula, Alcon) to prevent corneal drying. Intravitreal injections were guided by the use of a Stemi 305 stereo microscope (Zeiss, Oberkochen, Germany). Briefly, the right eye was slightly proptosed by periocular pressure and a 33G ½” needle with a 10 to 12° bevel fitted to a Hamilton micro syringe (Hamilton, Reno, NV, USA) was inserted just below the limbus at a ∼45° angle. Two microliters of rAAV (7.6 × 109 viral genomes) was slowly injected into the vitreous over the course of ∼1 minute. For the left eye, the same injection procedure was followed, but using vehicle (Hanks' Balanced Salt Solution with 0.14% Tween [HBSS-T]). Postinjection, a drop of GenTeal and a small amount of AK-POLY-BAC antibiotic ointment (Akorn, Lake Forest, IL, USA) were applied to the eye. Forty-eight hours after injection, mice were macroscopically inspected and those with any apparent ocular damage (e.g., cloudy eye) were not used for subsequent experiments.
10
Protonemal Growth and Stress Resistance in Physcomitrella
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Physcomitrella patens subsp. patens (Physcomitrium patens “Gransden 2004”, Freiburg) protonemata were grown on BCD medium supplemented with 5 mM ammonium tartrate (BCDAT) and/or 0.5% glucose with 1.5% agar (Helicon, Moscow, Russian Federation) in a Sanyo Plant Growth Incubator MLR-352H (Panasonic, Osaka, Japan) with a photon flux of 61 μM/m2•s during a 16-hour photoperiod at 24°C in 9 cm Petri dishes (Nishiyama et al., 2000). For proteomic and qRT-PCR analyses, the protonemata were grown in liquid BCDAT medium and collected on day 7. The gametophores were grown on free-ammonium tartrate BCD medium under the same conditions, and 8-week-old gametophores were used for analysis. For analysis of abiotic stress resistance, protonemata were grown on BCD medium without ammonium tartrate supplemented with 150 mM NaCl or 2 μM paraquat (PQ). For PpRALFs expression analysis, 7-day-old protonemata were treated with 100 mM hydrogen peroxide for 2 hours.
For morphological analysis, protonema tissue 2 mm in diameter was planted on 9 cm Petri dishes on BCD and BCDAT media.
For growth rate measurements, photographs were taken at day 30 of subcultivation. Protonemal tissues and cells were photographed using a Microscope Digital Eyepiece DCM-510 attached to a Stemi 305 stereomicroscope (Zeiss, Germany).
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Physcomitrella patens subsp. patens (Physcomitrium patens “Gransden 2004”, Freiburg) protonemata were grown on BCD medium supplemented with 5 mM ammonium tartrate (BCDAT) and/or 0.5% glucose with 1.5% agar (Helicon, Moscow, Russian Federation) in a Sanyo Plant Growth Incubator MLR-352H (Panasonic, Osaka, Japan) with a photon flux of 61 μM/m2•s during a 16-hour photoperiod at 24°C in 9 cm Petri dishes (Nishiyama et al., 2000). For proteomic and qRT-PCR analyses, the protonemata were grown in liquid BCDAT medium and collected on day 7. The gametophores were grown on free-ammonium tartrate BCD medium under the same conditions, and 8-week-old gametophores were used for analysis. For analysis of abiotic stress resistance, protonemata were grown on BCD medium without ammonium tartrate supplemented with 150 mM NaCl or 2 μM paraquat (PQ). For PpRALFs expression analysis, 7-day-old protonemata were treated with 100 mM hydrogen peroxide for 2 hours.
For morphological analysis, protonema tissue 2 mm in diameter was planted on 9 cm Petri dishes on BCD and BCDAT media.
For growth rate measurements, photographs were taken at day 30 of subcultivation. Protonemal tissues and cells were photographed using a Microscope Digital Eyepiece DCM-510 attached to a Stemi 305 stereomicroscope (Zeiss, Germany).
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