Males were moved to plates without hermaphrodites 1–2 days prior to imaging to maximize sperm retention. Males were soaked in 2 ml of 1× Sperm Buffer (50 mM HEPES, 25 mM KCl, 35 mM NaCl, 1 mM MgSO4, 5 mM CaCl2, 10 mM dextrose/D-glucose, pH 7.8, filter sterilized) with 0.5 μl of 5 μg/ml DAPI for 5 min in a crystallization dish, then transferred to poly-L-lysine coated slides. 2 mM levamisole was added, and worm tails were cut with a 17-gauge needle to release sperm/germline. Samples were imaged immediately using Sequential Scan Mode on a Leica DMi8 TCS SP8 confocal microscope using a 63×/1.40 HC PL APO CS2 oil objective.
Dmi8 tcs sp8 confocal microscope
Manufactured by Leica
Sourced in Germany
The Leica DMi8 TCS SP8 confocal microscope is a high-performance imaging system designed for advanced research applications. It features a modular design that allows for customization and integration of various detection technologies. The core function of this microscope is to provide high-resolution, optical sectioning of samples, enabling detailed analysis of cellular and subcellular structures.
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4 protocols using dmi8 tcs sp8 confocal microscope
1
Sperm Visualization and Extraction Protocol
2
Visualizing GSK-3β Phosphorylation in Mouse Brain
3
Immunofluorescence Analysis of Calreticulin Localization
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Cells grown on sterile cover slips in 6-well plates for 24 h and treated with DX and/or TG for an additional 16 h before being washed with warm PBS and fixed with 4% w/v paraformaldehyde in PBS for 20 min were blocked in 5% w/v sterile BSA in PBS for 10 min and then incubated with (a) antibodies to detect endogenous CRT released from cells that binds to the cell surface, or (b) cells were incubated with exogenous CRT and then examined by immunofluorescence. Briefly, adherent cells were treated with anti-human CRT (rabbit polyclonal, Thermofisher A3-900; 1:200) for one hour. For endogenous CRT detection, cells were then washed and incubated with Alexa Fluor® 488 conjugated secondary antibody (goat anti-rabbit IgG, Abcam-150077; 1:2000) or goat-anti Rab CY5 –Abcam-6564) for 30 min. Cells were washed again and the coverslip containing cells inverted onto a slide with a drop of ProLong® Gold anti-fade mounting medium with DAPI (Invitrogen). For exogenous CRT binding, cells were incubated for 30 min in the dark at RT. Digital images of cells were captured either on a Leica DM4000 B LED fluorescent microscope or Leica DMi8 TCS SP8 Confocal microscope at × 10, × 20 and × 40 magnification using LAS X digital software.
4
Confocal Imaging of Corpus Callosum
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