Inverted wide field microscope

Manufactured by Zeiss

The Inverted wide-field microscope is a laboratory instrument designed for observing and analyzing samples from below. It features a vertically oriented objective lens and a horizontally positioned sample stage, allowing for convenient examination of cell cultures and other transparent specimens.

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Lab products found in correlation

α ly6g 1a8, by Abcam (2 mentions) Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions) Rat igg2b anti mouse cd16 cd32 receptor antibody, by BD (1 mentions) Matlab, by MathWorks (1 mentions) Ix81 microscope, by Yokogawa (1 mentions) Axiocam mrm, by Zeiss (1 mentions) Alexa fluor 488 or 594, by Thermo Fisher Scientific (1 mentions) Axiovision release 4, by Zeiss (1 mentions) Donkey anti rabbit 488, by Thermo Fisher Scientific (1 mentions) Donkey anti mouse alexa fluor 555, by Thermo Fisher Scientific (1 mentions)

5 protocols using inverted wide field microscope

Quantifying Ly6G+ Neutrophils in Lung Cryosections

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Lungs were inflated with 1 ml 50% OCT (VWR) diluted in PBS and frozen on dry ice. Ten micrometers lung cryosections were cut using a cryostat (Bright OTF5000 LS) and sections were fixed in acetone for 10 min at room temperature. Sections were rehydrated twice in PBS before blocking with purified rat IgG2b anti–mouse CD16/CD32 receptor antibody (BD) to block Fc binding in FACS buffer for 30 min at room temperature. To detect Ly6G⁺ neutrophils, sections were stained with α-Ly6G 1A8 (1:800, Abcam) overnight at 4 °C. Sections were then washed twice in PBS and stained with a species-specific secondary antibody conjugated to Alexa Fluor 647 (1:200, Abcam) for 2 h in the dark at 4 °C. Sections were washed twice in PBS and coverslips were mounted into glass slides with ProLong® Gold Anti-fade Mountant with DAPI (ThermoFisher Scientific). The Zeiss Inverted Widefield Microscope on a 20× dry lens was used for obtaining images of the cells. Analysis of the images was performed on Fiji, an open-source imaging processing software (ImageJ).

Kirsebom F.C., Kausar F., Nuriev R., Makris S, & Johansson C. (2019). Neutrophil recruitment and activation are differentially dependent on MyD88/TRIF and MAVS signaling during RSV infection. Mucosal Immunology, 12(5), 1244-1255.

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Neutrophil Quantification in Lung Cryosections

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Lungs were inflated with 1ml 50% OCT (VWR) diluted in PBS and frozen on dry ice. Ten micrometers lung cryosections were cut using a cryostat (Bright OTF5000 LS) and sections were fixed in acetone for 10 min at room temperature. Sections were rehydrated twice in PBS before blocking with purified rat IgG2b anti–mouse CD16/CD32 receptor antibody (BD) to block Fc binding in FACS buffer for 30 min at room temperature. To detect Ly6G⁺ neutrophils, sections were stained with α-Ly6G 1A8 (1:800, Abcam) overnight at 4 °C. Sections were then washed twice in PBS and stained with a species-specific secondary antibody conjugated to Alexa Fluor 647 (1:200, Abcam) for 2 h in the dark at 4 °C. Sections were washed twice in PBS and coverslips were mounted into glass slides with ProLong^® Gold Anti-fade Mountant with DAPI (ThermoFisher Scientific). The Zeiss Inverted Widefield Microscope on a 20× dry lens was used for obtaining images of the cells. Analysis of the images was performed on Fiji, an open-source imaging processing software (ImageJ).

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Microscopy Imaging of Immunostained Samples

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Immunostained samples were imaged on an Olympus IX81 microscope equipped with a spinning disk module (Yokogawa) and back illuminated EMCCD (Andor Technology, iXon Ultra 888) with an exposure time of

100 m s

and EM gain of 250. A confocal z-stack of immunostained samples was acquired using a

60 \times

silicon immersion objective with

0.47 μ m

z-spacing.
Adult wings were imaged on an inverted wide field microscope (Zeiss) using a

5 \times

objective. The images were analyzed using a custom code written in MATLAB (Mathworks, USA) to measure wing area.

Dye N.A., Popović M., Iyer K.V., Fuhrmann J.F., Piscitello-Gómez R., Eaton S, & Jülicher F. (2021). Self-organized patterning of cell morphology via mechanosensitive feedback. eLife, 10, e57964.

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Intracellular Parasite Immunofluorescence Assay

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Immunofluorescence assays (IFAs) were performed using standard procedures. Briefly, intracellular parasites were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100–phosphate-buffered saline (PBS), and blocked in 3% bovine serum albumin (BSA)–PBS. Specified primary antibodies were diluted in 3% BSA–PBS, decorated onto blocked slides, and detected with antibodies conjugated to Alexa Fluor 594 or 488 (Molecular Probes). Parasites were then imaged on a Zeiss inverted wide-field microscope equipped with AxioCam MRM and AxioVision (release 4.8) with Deconvolution software.

Williams M.J., Alonso H., Enciso M., Egarter S., Sheiner L., Meissner M., Striepen B., Smith B.J, & Tonkin C.J. (2015). Two Essential Light Chains Regulate the MyoA Lever Arm To Promote Toxoplasma Gliding Motility. mBio, 6(5), e00845-15.

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FLG Effects on Adipocyte Lipid Droplets

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SGBS adipocytes were grown and allowed to differentiate for 14-days on cover slips. To study the effects of FLG treatment on lipid droplets, FLG at 10ng/ml was added to the culture media for 3 hours. Afterwards the cells were washed twice with PBS and fixed for 15 min with 4% PFA-PBS, permeabilized with 0.5% Triton-X for 5 min, blocked 1 hour with 5% donkey serum and thereafter incubated with primary antibody over night at 4°C. Immunolabeling was performed using rabbit polyclonal antibody against TLR5 (Pierce, Appleton, WI, USA, 1:50 in 1% donkey serum) and mouse monoclonal antibody against perilipin (Progen, Heidelberg, Germany, 1:200 in 1% donkey serum). As secondary antibodies donkey anti-mouse Alexa Fluor 555 and donkey anti-rabbit 488 (Invitrogen) were used. The labeled cells were imaged using an inverted wide-field microscope (Carl Zeiss) with a confocal unit and 40× oil/1.4 NA objective (Carl Zeiss). To count the percentage of cells in which lipid droplets were degraded, 200 control and FLG-treated cells were observed in randomly selected fields using 63× oil/1.4 NA objective.

Munukka E., Wiklund P., Partanen T., Välimäki S., Laakkonen E.K., Lehti M., Fischer-Posovzsky P., Wabitsch M., Cheng S., Huovinen P, & Pekkala S. (2016). Adipocytes as a Link Between Gut Microbiota-Derived Flagellin and Hepatocyte Fat Accumulation. PLoS ONE, 11(4), e0152786.

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