In order to assess the formation of microgametes after artificial removal of the EM, mature NF54 WT and PPLP2(−) gametocytes were incubated either with 0.05 mg ml−1 equinatoxin II (kindly provided by G. Anderluh, University of Ljubljana) in culture medium or in culture medium alone (control) for 10 min at 37°C. After washing with culture medium the gametocytes were activated by 100 μM XA at RT. At 20 min p.a. samples were taken and subjected to IFA. The sexual stage parasites were highlighted by Pfs230-labelling, followed by staining of nuclei as described above. The numbers of microgametes in relation to all Pfs230-positive parasites were counted at 630-fold magnification in 30 optical fields in triplicate using a Zeiss Axiolab fluorescence microscope.
Axiolab fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The Axiolab fluorescence microscope is a high-performance optical instrument designed for fluorescence imaging and analysis. It provides a reliable and accurate platform for researchers and scientists to observe and study fluorescently labeled samples. The core function of the Axiolab is to enable the visualization and analysis of fluorescent signals within a specimen, allowing for the investigation of various biological and chemical processes.
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Lab products found in correlation
Progres c12 digital camera, by Jenoptik (3 mentions)
Dabco, by Merck Group (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Antifade solution, by Thermo Fisher Scientific (1 mentions)
Anodisc filter, by Cytiva (1 mentions)
Anti α syn, by BioLegend (1 mentions)
Anti β tubulin 3, by Merck Group (1 mentions)
Alexa488 conjugated avidin, by Thermo Fisher Scientific (1 mentions)
To pro 3 iodide, by Thermo Fisher Scientific (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Anti ck18, by Abcam (1 mentions)
Anti β3 tubulin, by Merck Group (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Ivis spectrum, by PerkinElmer (1 mentions)
12 protocols using axiolab fluorescence microscope
1
Quantifying Microgamete Formation in Malaria Gametocytes
2
Quantifying Food Intake in C. elegans
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To study the effect of C. chinensis and E. ulmoides on the food intake of C. elegans, the E. coli strain OP50-GFP (CGC, Minneapolis, MN, United States) was used according to Raizen et al., (2012) (link). Extract-treated and control worms were transferred on the 7th and 12th day of adulthood to small agar plates seeded with a confluent lawn of OP50-GFP, and were allowed to feed for 15 min. Thereafter, about 25–30 nematodes per age and treatment were rinsed, washed in M9 buffer, moved to a 2% agarose pad on a glass slide, and anesthetized by 1 M NaN3. The worms were photographed with an Axiolab fluorescence microscope (Carl Zeiss, Jena, Germany) equipped with a GFP filter and a ProgRes C12 digital camera (Jenoptik, Jena, Germany). Mean fluorescence intensities per single worm were quantified using the CellProfiler software (Mcquin et al., 2018 (link)). The intensity values were normalized by subtracting the green autofluorescence values measured in extract-treated and control worms of the same age, fed with standard OP50.
3
Staining and Imaging Viral Particles
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Staining of the samples was carried out as described by Patel et al.26 (link). Briefly, all samples were diluted appropriately with 0.02 µm filtered 1 × TE buffer (pH 7.5, AppliChem, Darmstadt, Germany) to a concentration of 107 particles mL–1. For environmental samples with lower concentrations, a volume of 10 mL was used.
Then, 1 mL of each diluted sample (10 mL of environmental samples) was passed through a 0.02 µm Anodisc filter (Whatman) in duplicates. After complete desiccation, the filter was stained using a drop of 2 × SYBR gold dye (Thermo Fisher) with the virus side up, and incubated at room temperature for 15 min in the dark. Stained filters were mounted on a glass slide with 20 µL antifade solution (Thermo Fisher). Slides were analyzed using an Axiolab fluorescence microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 488 nm laser. A camera was used to take ten pictures per sample, which were analyzed using ImageJ (version 1.50i). Numbers of particles on the whole filter were calculated by multiplying the counts with the quotient of the area of the filter by area of the pictures.
Then, 1 mL of each diluted sample (10 mL of environmental samples) was passed through a 0.02 µm Anodisc filter (Whatman) in duplicates. After complete desiccation, the filter was stained using a drop of 2 × SYBR gold dye (Thermo Fisher) with the virus side up, and incubated at room temperature for 15 min in the dark. Stained filters were mounted on a glass slide with 20 µL antifade solution (Thermo Fisher). Slides were analyzed using an Axiolab fluorescence microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 488 nm laser. A camera was used to take ten pictures per sample, which were analyzed using ImageJ (version 1.50i). Numbers of particles on the whole filter were calculated by multiplying the counts with the quotient of the area of the filter by area of the pictures.
4
Fluorescent Staining of Cells
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Cells were stained with 10 μL each of 20 μg/mL AO (AO; Invitrogen, USA) and 20 μg/mL PI (PI; Invitrogen, USA). Then, 10 μL of the mixture were then loaded onto a microscope slide and observed under an Axiolab fluorescence microscope (Zeiss, Germany).
5
Immunocytochemical Analysis of Neural Samples in PD
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NS samples collected from AN and MT of 18 patients with PD were processed for immunocytochemistry. Slides were incubated overnight at 4 °C with primary anti-β-tubulin III (1:400; T2200 Sigma), anti-α-syn (1:400; BioLegend) and anti-phospho-α-syn (1:1000; Invitrogen, Waltham, MA) antibodies. On the next day, the slides were washed and incubated for 1 h at room temperature with Alexa Fluor-conjugated secondary antibodies (1:1000, Life Technologies, Waltham, MA). After washing, the slides were incubated with DAPI (1:2000) for 5 min and mounted with DABCO (Sigma-Aldrich, Milan, Italy). Images were acquired using the Axiolab fluorescence microscope (Zeiss, Oberkochen, Germany).
6
Quantifying Oxidative DNA Damage via Comet Assay
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In order to determine the endogenous (background) levels of oxidatively induced DNA damage we performed the sensitive technique of single cell gel electrophoresis (SCGE; Comet assay) under alkaline (denaturing) conditions as previously described [67 (link)]. We also used as a DNA damage probe the human repair enzyme OGG1 (New England Biolabs) to detect the specific presence of 8-oxo-dGuanine (8-oxo-dG) [24 (link)]. An increase in the tail moment (TM) suggests higher levels of oxidative DNA damage (primarily oxidized purines). Cells were observed under a Zeiss Axiolab fluorescence microscope equipped with a monochrome CCD camera. Analysis was conducted with Cometscore software (Tritek). All experiments were performed five times.
7
Autofluorescence Imaging in C. elegans
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The intestinal autofluorescence in extract-treated C. elegans was measured on the 7th and 12th days of adulthood according to Pincus et al., (2016) (link). Approximately 25 individuals per age and treatment were mounted on a 2% agarose pad on a microscope glass slide, and immobilized using 1 M sodium azide. Red autofluorescence was determined and imaged using the Axiolab fluorescence microscope (Carl Zeiss, Jena, Germany) with a TRITC filter set (excitation: 546 nm; emission: 600 nm), and equipped with a ProgRes C12 digital camera (Jenoptik, Jena, Germany). Mean fluorescence intensities per nematode were quantified densitometrically using the CellProfiler software (Mcquin et al., 2018 (link)).
8
Quantification of 8-oxo-dG in Cells
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A previously described assay based on the property of avidin to bind with high specificity to 8-oxo-dG was used for the 8-oxoG measurements [24 (link)]. Briefly, cells were fixed in methanol at −20 °C for 20 min and incubated for 15 min in TBS, 0.1 % Triton X-100. Blocking was performed in 15 % FBS, 0.1 % Triton X-100 in TBS for 2 h at room temperature (RT). Cells were then incubated with 10 μg/ml Alexa488-conjugated avidin (Invitrogen) in blocking solution for 1 h at 37 °C. Next they were rinsed twice in TBS, 0.1 % Triton X-100 for 5 min each round at room temperature. After a quick rinse in distilled water, DNA was counterstained with ToPro3-Iodide (LifeTechnologies) for 15 min at room temperature, followed by a final rinse in TBS.
Coverslips were mounted with ProLongGold (Invitrogen) and cells were observed under a Zeiss Axiolab fluorescence microscope equipped with a monochrome CCD camera. Analysis was conducted with NIH-imageJ, with respect to mean intensity in the nucleus (To-Pro3 served as a DNA reference). All experiments were repeated five times.
Coverslips were mounted with ProLongGold (Invitrogen) and cells were observed under a Zeiss Axiolab fluorescence microscope equipped with a monochrome CCD camera. Analysis was conducted with NIH-imageJ, with respect to mean intensity in the nucleus (To-Pro3 served as a DNA reference). All experiments were repeated five times.
9
Immunocytochemistry Analysis of Neural Markers
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Nasal swabs were also processed for immunocytochemistry. Slides were incubated overnight at 4°C with primary antibodies: anti‐β3 tubulin (1:400; catalog number [cn] T2200; Sigma), anti‐β3 tubulin (1:400; cn 322600; Invitrogen), anti‐β4 tubulin (1:500; cn T7941; Sigma), anti‐CK18 (1:300; cn ab32118; Abcam), anti‐p62 (1:150; cn 610833; BD), anti‐PGP9.5 (1:600; cn GTX634797; Genetex), anti‐TARDBP (1:200; cn sc‐376311; Santa Cruz), anti‐TDP‐43 C‐term (1:200; cn T1580; Merck), anti‐TDP‐43 pS409/410 (1:800; cn TIP‐PTD‐P01; Cosmobio)
22 (link) antibodies. The next day, slides were washed and incubated for 1 hour at room temperature with Alexa Fluor‐conjugated secondary antibodies (1:1000, Life Technologies). After washing, slides were incubated with DAPI (1:2000) for 5 minutes and mounted with DABCO (Sigma‐Aldrich). Images were acquired using an Axiolab fluorescence microscope (Zeiss).
22 (link) antibodies. The next day, slides were washed and incubated for 1 hour at room temperature with Alexa Fluor‐conjugated secondary antibodies (1:1000, Life Technologies). After washing, slides were incubated with DAPI (1:2000) for 5 minutes and mounted with DABCO (Sigma‐Aldrich). Images were acquired using an Axiolab fluorescence microscope (Zeiss).
10
Quantifying Endogenous ROS in C. elegans
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The strain JV1, which expresses the YFP-based hydrogen peroxide sensor “HyPer” was used to ascertain the endogenous ROS level according to Back et al. [36 (link)]. On the 12th day of adulthood, about 25 individuals per treatment group were transferred to a 2% agarose pad on a microscope glass slide, and immobilized using 1 M sodium azide. The Axiolab fluorescence microscope (Carl Zeiss, Jena, Germany) equipped with a YFP filter set, a ProgRes C12 digital camera (Jenoptik, Jena, Germany), and an objective with 10× magnification was used to image the worms. Mean fluorescence intensities per single worm were quantified using the CellProfiler software [37 (link)]. The intensity values were normalized by subtracting the yellow autofluorescence values measured in extract-treated and control worms of the same age. DMSO-treated worms exposed to 10 mM H2O2 for 30 min on day 12 of adulthood were used as a positive control. Three independent experiments were performed per treatment group.
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