Bx4 microscope

The different CNTs were characterized by scanning electron microscopy using a JEOL SEM, model JSM-5800 LV.
Raman spectroscopy was performed using a micro-Raman LabRAM HR (Horiba Jobn Yvon, Piscataway, NJ, USA) coupled to an Olympus BX-4 microscope (Olympus, Miami, FL, USA) and Spectrum Gx (Perkin Elmer, Waltham, MA, USA). The sample was excited using a laser line at a wavelength of 632.8 nm, and all measurements were performed at room temperature.
The functional groups present in different CNTs were determined by Fourier Transform Infrared (FTIR) spectroscopy in transmittance mode using a Carry 600 Series FTIR Spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with a zinc selenide accessory in attenuated total reflectance (ATR) mode; the wavenumber ranged of 600 cm⁻¹ to 4000 cm⁻¹. CNTs were deposited as dry powders onto a zinc selenide (ZnSe) window, no solvents were used in this process, and all determinations were made at room temperature.
A thermogravimetric analysis (TGA) was performed using a SDT Q600 V20.9 Build 20 (TA Instruments, New Castle, DE, USA) heated at a rate of 10 °C/min to 1000 °C, and air was introduced into the samples at a rate of 25 mL/min.
Finally, 10% SDS–PAGE was carried out, and the proteins were visualized using the ProteoSilver™ Silver Stain Kit (Sigma–Aldrich) to demonstrate the presence of proteins in the sample.

5 µm lung sections were incubated in DNPH solution in the dark for 30 min and then blocked for 30 min at room temperature. The sections were then incubated with primary antibody (1:100) at 4 °C overnight and were then incubated with biotinylated secondary antibody for 1 h. After being incubated with the streptavidin-conjugated HRP for 30 min, the slides were incubated with the DAB-A/B mixture. The slides were examined using a Zeiss Axio Observer 7 microscope (Zeiss, Oberkochen, Germany) or an Olympus BX4 microscope (Watford, UK) under a constant exposure level.

To assess the microglial activation, oxidative response and demyelination in the CNS, treated and untreated groups of C57BL/6 mice were euthanized at the chronic stage of EAE disease (day 30) by CO₂ asphyxiation. Their spinal cords of the lumbar region were removed and fixed in 10% buffered formalin. After paraffin embedding, transverse sections of spinal cord of 12 μm-thick (nine sections per animal) were stained with Iba-1, iNOS, and NF-H, respectively. The spinal cord sections from five representative mice (BCP treated and untreated) were processed and stained with Hoechst 33342 dye (for nuclei stain). A Q imaging digital camera connected to an Olympus Bx4 microscope was used. The immunostaining was assessed at four levels of the lumbar spinal cord. Specifically, four alternate 5-μm sections of the lumbar spinal cord with an individual distance of 150 μm were obtained between L4 and L6. A threshold optical density that best discriminated staining from the background was obtained using the NIH ImageJ 1.36b imaging software (NIH, Bethesda, MD, USA). We captured four images of each spinal cord subregion (dorsal, ventral, lateral, central) per section (eight images per section and 32 images per mouse, n = 5 animals/group). Then, the total pixel intensity was determined and the data were expressed as optical density (O.D.).