To analyze microglia activation upon TiLV infection, the transgenic Tg(mpeg1.1:mCherryF)ump2 zebrafish larvae infected with TiLV or mock-infected, were anesthetized at 24 and 48 hpi, and positioned dorsally in 1% low-melting point agarose (Sigma-Aldrich) in E3 w/o methylene blue on a glass bottom dish filled with E3 medium containing 0.1 mg/ml of MS-222 (Sigma-Aldrich). Midbrain region of each larvae was imaged with the Zeiss LSM 900 Airyscan 2 confocal microscope using the C-Apochromat 40x/NA 1.2 water objective. For image analysis, ImageJ/Fiji was used. Maximum intensity projections were created, and fluorescence photomicrograph were converted to representative binary images using previously described protocol to quantify microglia morphology (33 (link)). Then, the circularity index (0–1) was analyzed as a 4-pi (area/perimeter^2) formula, where the value of 1.0 indicates a perfect circle. Each image was additionally manually verified for the correct microglia outliers and any artifacts rejected from the analysis.
C apochromat 40x na 1.2 water objective
Manufactured by Zeiss
The C-Apochromat 40x/NA 1.2 water objective is a high-performance microscope objective lens produced by Zeiss. It features a magnification of 40x and a numerical aperture of 1.2, making it well-suited for applications requiring high-resolution imaging and light collection in aqueous environments.
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Lab products found in correlation
Low melting point agarose, by Merck Group (1 mentions)
Ms 222, by Merck Group (1 mentions)
Lsm 900 airyscan 2 confocal microscope, by Zeiss (1 mentions)
E cadherin, by Proteintech (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Vimentin, by Developmental Studies Hybridoma Bank (1 mentions)
2 protocols using c apochromat 40x na 1.2 water objective
1
Quantifying Microglia Activation in TiLV-infected Zebrafish
2
Immunofluorescent Staining of Adherent Cells
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The adherent cells were fixed with 3.7% formaldehyde/PBS for 15 min at RT, followed by a 15 min permeabilisation step in 0.2% Triton X-100 at RT. The samples were then blocked with 3% BSA/PBS for 1h at RT. The cells were then stained overnight at 4 • C with 1:40 Vimentin (PN:AMF-17b, Developmental Studies Hybridoma Bank, Iowa City, IA) or 1:100 E-Cadherin (PN:60335-1-lg, Proteintech) primary antibodies. The samples were then treated with 1:1000 secondary Alexa Fluor 488 (PN:A32723, Invitrogen) and 5 µg/mL DAPI in 3% BSA/PBS solution for 2 h at RT. Images were recorded using a Zeiss LSM700 confocal microscope of the CMCB light microscopy facility, incorporating a Zeiss C-Apochromat 40x NA 1.2 water objective.
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