Anesthetized mice were locally injected with either Taipoxin or O. scutellatus venom close to soleus muscles. Muscles were dissected at different time points, fixed in 4% paraformaldehyde in PBS for 30 min at room temperature, and quenched in 0.24% NH4Cl PBS for 20 min. After permeabilization and 2 h saturation in blocking solution (15% goat serum, 2% BSA, 0.25% gelatine, 0.20% glycine, 0.5% Triton X-100 in PBS), samples were incubated with primary antibodies for 72 h in blocking solution at 4°C. Muscles were then washed in PBS and incubated with secondary antibodies diluted in PBS+0.5% Triton X-100. Images were collected with a Leica SP5 confocal microscope equipped with a 40× HCX PL APO NA 1.4. Laser excitation line, power intensity and emission range were chosen according to each fluorophore in different samples to minimize bleed-through.
Hcx pl apo na 1
Manufactured by Leica
The 40× HCX PL APO NA 1.4 is a high-performance objective lens designed for Leica microscopes. It features a 40× magnification and a numerical aperture of 1.4, providing high-resolution imaging capabilities.
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4 protocols using hcx pl apo na 1
1
Investigating Neurotoxin Effects on Muscle
2
Confocal Microscopy Imaging Protocol
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Samples from both neuronal culture and in vivo experiments were analyzed with a Leica SP5 Confocal microscope with a 40× HCX PL APO NA 1.4 oil immersion objective. Laser excitation line, power intensity, and emission range were chosen to minimize signal bleed-through. Laser excitation line, power intensity, and emission range were chosen according to each fluorophore in different samples to minimize bleed-through. Raw images were processed with ImageJ without altering the intensity of the signals.
3
Spindle Orientation Imaging Protocol
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Samples were acquired from the tissue apical surface with a 40× oil immersion objective (HCX PL APO NA 1.25; Leica) plus 3× zoom, on an inverted confocal microscope (TCS SP5; Leica). Z-views were generated in ImageJ. Spindle orientation measurements were performed using the angle tool of ImageJ on the Z-view images, taking the apical membrane as a reference plane.
4
Ratiometric Live-Cell FRET Imaging
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Fluorescence measurements were performed in a Leica TCS-SP5 laser scanning confocal microscope equipped with resonant scanners and hybrid photon detectors (HyD) and using a 40x objective (HCX PL APO, NA 1.25, oil-immersion). Cells were excited with a UV diode (405 nm) and emission was detected at 450–490 nm (CFP) and 520–590 nm (FRET). Pictures were acquired every 60 sec. After a baseline of 5 min, media containing metabolites or not were added and changes in fluorescence were recorded for 15 minutes. During imaging, cells were maintained at 37 °C and 5% CO2. Image processing was performed with ImageJ (http://fiji.sc/ ). After background subtraction and registration, single cells were segmented by threshold and mean intensity was measured for each channel at every time point. The ratio was calculated by dividing the mean intensity of the acceptor (FRET) by the intensity of the donor (CFP). Ratios calculated from every cell were normalized to baseline measurements.
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