CalipHluor trafficking in coelomocytes was done in transgenic strains expressing endosomal markers such as GFP::RAB-5 (EE), GFP::RAB-7 (LE) and LMP-1::GFP (Ly) as described previously by our lab 21 (link). Briefly, worms were injected with CalipHluorA647 (500 nM) and incubated for specific time points and transferred on to ice. Worms were anaesthetized using 40 mM of sodium azide in M9 solution. Worms were then imaged on Leica TCS SP5 II STED laser scanning confocal microscope (Leica Microsystems, Inc., Buffalo Grove, IL) using an Argon ion laser for 488 nm excitation and He-Ne laser for 633nm excitations with a set of filters suitable for GFP and Alexa 647 respectively. Colocalization of GFP and CalipHluorA647 was determined by counting the number CalipHluorA647 positive puncta that colocalize with GFP-positive puncta and quantified as a percentage of total number of CalipHluorA647 positive puncta21 (link). In order to confirm lysosomal labeling in a given genetic background, the same procedure was performed on the relevant mutant or RNAi knockdown in pwIs50 [lmp-1::GFP + Cb-unc-119(+)].
Tcs sp5 2 sted laser scanning confocal microscope
Manufactured by Leica
Sourced in United States
The Leica TCS SP5 II STED laser scanning confocal microscope is a high-performance imaging system designed for advanced fluorescence microscopy. It combines the capabilities of confocal laser scanning microscopy with the super-resolution imaging technique of Stimulated Emission Depletion (STED) microscopy. The system features a modular and flexible design, allowing for customization to meet specific research requirements.
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4 protocols using tcs sp5 2 sted laser scanning confocal microscope
1
Visualizing Endosomal Trafficking of CalipHluor
2
Temporal Mapping of I-switch and Clensor
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Temporal mapping of I-switch and Clensor was done in 10 worms of pwIs50 [lmp-1::GFP + Cb-unc-119(+)] as previously described by our lab (Surana et al., 2011 (link)). Briefly, worms were injected with 500 nM of I4cLYA647 or ClensorA647, incubated at 22°C for 1 hr, and then imaged using Leica TCS SP5 II STED laser scanning confocal microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA). Colocalization of GFP and I4cLYA647 or ClensorA647 was determined by counting the numbers of Alexa647 positive puncta that colocalize with GFP positive puncta and expressing them as a percentage of the total number of Alexa 647 positive puncta. In order to confirm lysosomal labeling in a given genetic background, the same procedure was performed on the relevant mutant or RNAi knockdown in pwIs50 [lmp-1::GFP + Cb-unc-119(+)].
3
Immunocytochemistry for Microscopy Analysis
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Cells grown on glass coverslips were fixed in 4% paraformaldehyde for 10 min, washed in PBS and incubated with 0.3% (v/v) Triton X-100 for 10 min at RT. Following, cells were washed in PBS and blocked with 10% AB serum in PBS for 30 min. Coverslips were then incubated overnight at 4 °C with primary antibodies, diluted in the same buffer. Cells then were washed and incubated with Alexa Flour®-488 secondary anti-rabbit and Alexa Flour®-555 secondary anti-mouse antibodies (Invitrogen) at RT for 60 min. After washing, coverslips were mounted onto glass slide in Mowiol (Calbiochem) with 4′,6-diamidino-2-phenylindole (DAPI) solution [10% (w/v) Moviol, 1 µg/ml DAPI]. Coverslips were examined on a Leica TCS SP5 II STED laser scanning confocal microscope (Leica Microsystems).
4
Immunofluorescence Staining of HeLa Cells
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HeLa cells were seeded in eight chamber microscope slides (Nunc 155409) and treated with siRNA as previously described. After 72 hr, cells were washed with PBS, then fixed with 4% paraformaldehyde (Sigma) in PBST (PBT +0.05% Tween-20), freshly prepared by heating at 65° C until clear, at RT for 15 min. Fixative was removed and chilled (−20°C) methanol was added dropwise and incubated at RT for 15 min. Cells were washed with PBS and blocked with 10% FBS in PBST at RT for 1 hr. Primary antibody incubation was performed in 10% FBS in PBST at 4°C overnight. After 4 × 5 min washes with PBST secondary antibody incubation was performed at RT for 1 hr. After 4 × 5 min washes with PBST, cells were treated with SlowFade Gold antifade reagent with DAPI (Molecular Probes) and imaged using a Leica TCS SP5 II STED laser scanning confocal microscope (Leica Microsystems, Inc.).
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