Eos digital camera

Stained fish skeletons were imaged using a Nikon SMZ18 stereomicroscope and NIS-Elements software. Adult fish were anesthetized with Tricaine mesylate (MS-222; 0.04%, Western Chemical Inc.) and photographed using a Canon EOS digital camera. Imaging of live and stained larval fish were performed using a Zeiss LSM 800 confocal microscope and ZEN Blue software or a Nikon SMZ18 stereomicroscope and NIS-Elements software for low magnification images of whole larvae. Larvae were anesthetized with MS-222 and embedded in 0.5% low-melting agarose for imaging. Confocal z-stacks were processed using ZEN blue software.

Following deparaffinization and hydration, sections were selected randomly from the lumbar spinal cord and hippocampus. All sections were retrieved by pressure cooker and permeabilized by 20% tween for 20 min for fluorescence immunostaining. The nonspecific area was blocked with 0.1% BSA in 0.1% Triton X-100/PBS for 20 min. The sections were incubated overnight at 4^◦ C with Anti-MBP antibody (1:500, Abcam [ab218011], USA) and Anti-GFAP antibody (1:800, Abcam [ab68428], USA) and Anti-IDO-1 Antibody (1:250, EMD Millipore [MABF850], Germany), as primary antibody, in 0.03% PBS-Triton- × 100 supplemented with 5% normal goat serum (Abcam[ab7481-], USA). The slides were incubated with appropriate secondary antibodies (Alexa Fluor 488 and 566 (1:500, Thermo Fisher Scientific, USA), and Hoechst (1:1000, Abcam [ab228550], USA) for 1 h. Samples were analysed using a fluorescent microscope (Olympus IX-71; Olympus, Tokyo, Japan) equipped with a Canon EOS digital camera. For quantification of plaque number, we used soteriological dissector method for particle number. after preparation of immunohistochemically imaging of spinal cord, two adjust section with mentioned distance compared them (position and plaque size). The plaque with different position were counted. Totally number of each spinal cord plaque calculated for comparison between groups. Area of plaque estimated image J software.

After 5 days, differentiated cultures were used for IF analysis. Cells in 12-well plates were fixed in 2% paraformaldehyde (PFA) for 20 min, washed with PBS, and permeabilized using 20% tween for 20 min, and then blocked in PBS, 2% triton, 5% horse serum (PBSTS) for 20 min. Cells were then incubated overnight with GFAP and MBP primary antibodies (Sigma Aldrich, USA) at 1:800 and 1:500 dilutions, respectively⁷⁸ . Wells were then washed in PBS and incubated for 1 hour with Alexa Fluor 488 or 568 (1:500 dilution) conjugated anti-mouse or anti-rabbit secondary antibodies, respectively. Representative pictures of each well (10–12 fields/well) were taken using a fluorescent microscope (Olympus IX-71) equipped with a Canon EOS digital camera. Cell counts were then performed and data presented as percent positive cells per treatment condition.

The sections were dewaxed, rehydrated, and then immersed into citrate buffer for antigen repair. After that, the sections were blocked with 2% bovine serum albumin and then incubated overnight at 4 °C with the following primary antibodies: anti-iNOS (1:200; Invitrogen, USA), anti-ionized calcium binding adaptor molecule-1 (Iba-1) (1:100, Abcam, USA), anti-Arg-1 (1:200, Santa Cruz Biotechnology, USA), followed by incubation with the secondary antibodies Alex Fluor® 488-labeled secondary antibody (green; 1:1000; Abcam), Alex Fluor® 647-labeled secondary antibody (red; 1:1000; Abcam) and Hoechst (blue; 1:1000) for 1 h at room temperature. Slides were observed by a fluorescent microscope (Olympus IX-71) equipped with a Canon EOS digital camera. The iNOS, Arg-1, and Iba-1 positive cells were separately counted using Image J software.

Differentiated cultures were then used for analysis by immunofluorescence (IF). Cells were fixed for 20 min in 2% paraformaldehyde (PFA) in 12-well plates followed by washing with PBS. Thereafter, cells were permeabilized for 20 min using 20% tween and then blocked for 20 min in PBS, 2% triton, 5% horse serum (PBSTS). Thereafter, cells were incubated overnight with monoclonal antibodies against myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and β-III tubulin (Sigma Aldrich, USA) at 1:500, 1:800 and 1:700 dilutions, respectively. Following several washes in PBS, wells were incubated for 1 h with Alexa Fluor 488 or 568 (1:500 dilution) conjugated anti-mouse or anti-rabbit secondary antibodies, respectively. Representative pictures of each well (10–12 fields/well) were taken using a fluorescent microscope (Olympus IX-71) equipped with a Canon EOS digital camera. Cells were then counted, and data presented as percent positive cells per treatment condition.

Cucumber fruits with a length of 0.2, 0.35, and 0.6 cm, as well as roots and receptacles, were fixed in FAA and vacuumed for 20 min then dehydrated through a gradient ethanol series (50, 60, 70, 85, 90, 95, and 100 % (v/v)) for every 5 min. They were then subjected to critical-point drying using the Leica EM CPD300 desiccator (Feica, Germany) and gold–palladium coating using the Leica EM SCD050 ion sputter and carbon coating unit (Feica, Germany). Finally, all samples were observed under an ALTO 1000 scanning electron microscope (Gatan, United States). Digital photos of cucumber fruits of different lengths were taken using a Canon EOS digital camera. Microscopy photos were taken using a LECIA DM 2500 microscope.

The sections were dewaxed, rehydrated, and then immersed into citrate buffer for antigen retrieval. After that, the sections were blocked with 2% bovine serum albumin and then incubated overnight at 4 °C with the following primary antibodies: anti-ionized calcium binding adaptor molecule-1 (Iba-1) (1:100; Abcam, Cambridge, MA, USA), anti-CD68 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD206 (1:200; Santa Cruz Biotechnology), anti-P2X7 (1:200; Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA), anti- microtubule-associated protein 2 (MAP2; 1:200; Abcam), anti-CD11b (1:200; Abcam), followed by incubation with the secondary antibodies Alex Fluor® 488-labeled secondary antibody (green; 1:1000; Abcam), Alex Fluor® 647-labeled secondary antibody (red; 1:1000; Abcam) and DAPI (blue; 1:1000; Santa Cruz Biotechnology) for 1 h at room temperature. Slides were observed by a fluorescent microscope equipped with a Canon EOS digital camera.