We generated transgenic lines expressing TIR-1 under its endogenous promoter by expressing bamEx102[tir-1p::tir-1b::mCherry::unc-54 3’UTR+ceh-22::gfp] in ABC16 (tir-1(qd4); oxIs12[unc-47p::GFP, lin-15(+)]) animals using standard microinjection (Mello et al., 1991 (link)). L4 stage animals were imaged on Perkin Elmer Precisely UltraVIEW VoX confocal imaging system. To determine the subcellular localization of TIR-1, we generated transgenic animals expressing unc-47p::mScarlet::tir-1b:: let-858 3’UTR and co-injection marker ceh-22::gfp in EG1285 (oxIs12[unc-47p::GFP, lin-15(+)]) animals. To determine the tissue specificity of TIR-1 function, we generated transgenic animals expressing unc-47p::mScarlet::tir-1b::let-858 3’UTR and co-injection marker pstr-1::gfp in ABC16 (tir-1(qd4); oxIs12 [unc-47p::GFP]) animals. To determine whether TIR-1 promotes chronic axon degeneration, we expressed unc-47p::tir-1b::mCherry::unc-54 3’UTR and co-injection marker pstr-1::gfp in EG1285 (oxIs12[unc-47p::GFP, lin-15(+)]) animals. Transgenic lines were established using standard protocols (Mello et al., 1991 (link)). L4 stage animals were imaged on Perkin Elmer Precisely UltraVIEW VoX confocal imaging system.
Precisely ultra view vox confocal imaging system
Manufactured by PerkinElmer
Sourced in United States
The Precisely Ultra VIEW VOX Confocal Imaging System is a high-performance microscope designed for advanced live-cell and fixed-sample imaging. It features a spinning disc confocal technology that enables rapid, high-resolution imaging with minimal photodamage to samples. The system is capable of acquiring 3D and 4D images with high spatial and temporal resolution.
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10 protocols using precisely ultra view vox confocal imaging system
1
TIR-1 Subcellular Localization and Function
2
Visualization of Gap Junction Transfer
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COCs isolated from ovaries of PMSG-primed female mice were microinjected with 5% Lucifer yellow (Molecular Probes, Eugene, OR) and rhodamine dextran for 1 min. Injections resulted in dye filling the oocyte and further spreading to the cumulus cells through gap junctions. Rhodamine dextran, which cannot pass the gap junction channels due to its high molecular weight, confirmed the spread of dye observed reflected transfer through gap junctions. The experiments were recorded with a Perkin Elmer precisely Ultra VIEW VOX Confocal Imaging System (PerkinElmer, Waltham, MA, USA).
3
Live Imaging of Meiotic Spindle Dynamics
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After microinjecting Spc24 siRNA and α-tubulin-GFP mRNA, oocytes were incubated for 24 h in M2 medium supplemented with IBMX. Microtubule and chromosome dynamics were filmed on a Perkin Elmer precisely Ultra VIEW VOX Confocal Imaging System. A narrow band passed EGFP and BFP filter sets and a 30% cut neutral density filter from Chroma. Exposure time was set ranging between 300–800 ms depending on the α-tubulin-GFP and Hoechst 33342 fluorescence levels. The acquisition of digital time-lapse images was controlled by IP Lab (Scanalytics) or AQM6 (Andor/Kinetic-imaging) software packages. Confocal images of spindles and chromosomes in live oocytes were acquired with a 20× oil objective on a spinning disk confocal microscope (Perkin Elmer).
4
ROS Level Detection in Oocytes
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Method for ROS level detection in oocytes was conducted as described in a previous study [34 (link)]. The procedure is briefly summarized as follows. Oocytes were collected at different aging times with or without NAC treatment, and then incubated for 30 min at 37°C in M2 supplemented with 10 mM carboxy-H2DCF diacetate (Cat #S0033, Beyotime). After washing in M2 medium for at least 3 times, oocytes were observed under a spinning disk confocal microscope (Perkin Elmer) with identical settings. Images were analyzed by a Perkin Elmer precisely Ultra VIEW VOX Confocal Imaging System. The fluorescence intensity analysis for each oocyte was conducted using ZEN (2012) software.
5
ROS Quantification in Oocytes
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ROS levels in oocytes were assessed according to the method previously described [20 (link)]. The procedure is briefly summarized as follows. Oocytes from different experimental conditions were processed at the same time. Oocytes were incubated for 30 min at 37°C in M2 supplemented with 10 mM carboxy-H2DCF diacetate (Cat #S0033, Beyotime). After washing, oocytes were observed under a spinning disk confocal microscope (Perkin Elmer) with identical settings. Images were analyzed by a Perkin Elmer precisely Ultra VIEW VOX Confocal Imaging System.
6
Live Cell Imaging of Oocyte Meiosis
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The GV oocytes were cultured for 3 h at 37 °C to allow oocytes to enter the GVBD stage, and then oocytes were cultured in M2 medium containing Hoechst‐33342 (to label DNA, b2261, Sigma‐Aldrich) and SiR‐Tubulin (to image tubulin, CY‐SC002, Cytoskeleton). The live oocytes were imaged using the PerkinElmer precisely Ultra VIEW VOX Confocal Imaging System (PerkinElmer, Waltham, MA, USA).[78
] The imaging process took place in a controlled environment with a 5% CO2 atmosphere at 37 °C.
] The imaging process took place in a controlled environment with a 5% CO2 atmosphere at 37 °C.
7
Visualizing Actin and Chromosome Dynamics during Oocyte Maturation
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After microinjecting Alexa 488-Phalloidin (1 μM) and WASH1 MO, oocytes were incubated in M2 medium that contained Hoechst 33342 (5 ng/ml; Sigma) to acquire images of actin and chromosome dynamics during oocyte maturation. Images for actin dynamics were acquired using a Perkin Elmer precisely Ultra VIEW VOX confocal Imaging System. The exposure time was set to between 200 and 800 ms. Digital time-lapse images were acquired under the control of IP Lab (Scanalytics) or AQM6 (Andor/Kinetic-imaging) software. Confocal images of actin in viable oocytes were acquired with a 10x objective with a spinning disk confocal microscope (Perkin Elmer).
8
Visualizing Microtubule and Chromosome Dynamics
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After microinjecting MAP4-eGFP mRNA and H2B-mCherry mRNA, oocytes were incubated for 1h in M2 medium. Microtubule and chromosome dynamics were filmed on a Perkin Elmer precisely Ultra VIEW VOX Confocal Imaging System. Oocytes were exposed once an hour for 10h, and then exposed every 30 min for an extra 7h. Confocal images were acquired with a 20x objective on a spinning disk confocal microscope.
9
Imaging Oocyte Microtubule and Chromosome Dynamics
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After microinjecting MAP4-eGFP mRNA and H2B-mCherry mRNA, oocytes were incubated for 1h in M2 medium. Microtubule and chromosome dynamics were filmed on a Perkin Elmer precisely Ultra VIEW VOX Confocal Imaging System. Oocytes were exposed once an hour for 10h, and then exposed every 20 min for an extra 12h. The acquisition of digital time-lapse images was controlled by IP Lab (Scanalytics) or AQM6 (Andor/Kinetic-imaging) software packages. Confocal images were acquired with a 20x oil objective on a spinning disk confocal microscope.
10
Dynamics of Cell Cycle Proteins in Oocytes
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CCNB1‐GFP, MAP4‐GFP, H2B‐mcherry dynamics was filmed on a Perkin Elmer precisely Ultra VIEW VOX Confocal Imaging System equipped with an incubator chamber (at 37°C, 5% CO2) filled with M2 medium covered with a layer of paraffin oil. The image was obtained by the Volocity 6.0 software. Septin 9 siRNA‐injected oocytes and control siRNA‐injected oocytes were incubated in M2 medium within 200 μM IBMX for 24 h (at 37°C, 5% CO2). And next we released the oocytes from M2 medium and prepared them for time‐lapse imaging. Before designing this experiment, we had set up a procedure with shooting every 30 min to track and record the expression changes for CCNB1‐GFP for 14 h and MAP4‐GFP, H2B‐mcherry for 14 h.
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