Ls 50b luminescence spectrophotometer

Scanning electron microscopy and energy-dispersive X-ray mapping analysis images were obtained using a JEOL JSM 6335F instrument, at an acceleration voltage of 10 kV and 22 kV, respectively. High resolution transmission electron microscopy images were obtained using a JEOL JEM 3000 F microscope at an acceleration voltage of 300 kV. An inverted optical microscope (Nikon Eclipse Instrument Inc. Ti-S/L100), coupled with 20× objective was used to track the speed of the micromotors. Fluorescence images were obtained using an Epi-fluorescence attachment with a UV-2E/C (DAPI) filter cube. UV-VIS experiments were carried out using a Perkin-Elmer Lambda 20 spectrophotometer. Fluorescence spectra were recorded at 25 °C with a PerkinElmer LS-50B luminescence spectrophotometer equipped with a Xe flash lamp. The excitation and emission slit widths were 5 nm and scan speed was 1000 nm min^–1.

The LP was assessed as a marker of oxidative damage, using lipid-fluorescence product formation as a marker. Forty-two animals were killed by decapitation 24 h after either SCI or sham surgery, when the peak of LP levels is reached, according to Diaz-Ruiz et al. [6 (link)]. The samples of wet tissue were taken in the same way as described above. Lipid-soluble fluorescent products (LFP), an index of LP, were measured according to the technique described by Triggs and Willmore [24 (link)] and modified by Santamaría et al. [25 (link)]. Briefly, each sample of spinal cord tissue was homogenized in 3 ml of saline solution (0.9% NaCl). One-milliliter aliquots of the homogenate were added to 4 ml of a chloroform-methanol mixture (2 : 1, v/v). After stirring, the mixture was ice-cooled for 30 min to allow phase separation and the fluorescence of the chloroform layer was measured in a Perkin-Elmer LS50B luminescence spectrophotometer at 370 nm (excitation) and 430 nm (emission). The sensitivity of the spectrophotometer was adjusted to 150 fluorescence units with a quinine standard solution (0.1 g/ml). The values were expressed as fluorescent units per gram of wet tissue.

Seventy-two hours after sham operation or contusion, animals were anesthetized and euthanized by cardiac perfusion with saline solution. Afterwards, 1 cm of spinal cord at the T9 level was dissected from every rat to measure lipid-soluble fluorescent products (LFP), which provide an index of LP according to a previously described method (14 (link)). The spinal cord fragments were homogenized in 3 ml of saline solution (0.9% NaCl). One-milliliter aliquots of the homogenate were added to 4 ml of a chloroform-methanol mixture (2:1, v/v). After stirring, the mixture was cooled with ice for 30 min to allow phase separation. The fluorescence of the chloroform layer was measured in a Perkin-Elmer LS50B Luminescence spectrophotometer at 370 nm of excitation and 430 nm of emission wavelengths. The sensitivity of the spectrophotometer was adjusted to 150 fluorescence units with a quinine standard solution (0.1 mg/mL). Results were expressed as fluorescence units/g tissue and evaluated by a blinded observer. Statistical analysis of the weights of spinal cord wet tissue of all samples yielded no significant differences between groups and experiments.

Scanning electron microscopy (SEM) measurements were performed using a Hitachi TM-1000 Tabletop microscope (Hitachi High-Technologies, Tokyo, Japan). The G dispersions were dried for a few days and then covered with a ~50 nm Au layer. The solid state samples were observed at different magnifications by means of an electron beam accelerated at 15 kV, under high vacuum. The dispersions were also observed by transmission electron microscopy (TEM) using a Zeiss EM-10C/CR instrument (Oberkochen, Germany) operating at a voltage of 60 kV.
Fluorescence spectra were recorded at 25 °C with a PerkinElmer LS-50B luminescence spectrophotometer equipped with a Xe flash lamp and quartz cuvettes of 1 cm path length thermostatised with a Thermomix BU bath. The excitation and emission slit widths were 5 nm and scan speed 1000 nm min⁻¹. The acquisition and data analysis were performed using the Perkin-Elmer FLwin Lab software.
Solutions were degassed using an ultrasonic bath (Selecta, Barcelona, Spain). A Hielscher UP400S ultrasonic tip (Hielscher Ultrasonics GmbH, Teltow, Germany) equipped with a titanium sonotrode with a diameter of 7 mm and length of 100 mm was used to prepare the G dispersions. Dispersions were centrifuged using an Orto Alresa Digicen refrigerated centrifuge (Madrid, Spain).