Confocal fluorescent microscope

Methanol (CH₃OH) was purchased from RCI Labscan, Thailand. Gallic acid monohydrate (C₇H₆O₅·H₂O) and sodium carbonate anhydrous (Na₂CO₃) were purchased from Riedel-de Haën, Germany. Folin-Ciocalteu's phenol reagent and ethanol (C₂H₅OH) were purchased from Merck, Germany. Thiazolyl Blue Tetrazolium Bromide (MTT), allantoin, 2′,7′-dichlorofluorescin diacetate, quercetin (C₁₅H₁₀O₇), aluminum chloride (AlCl₃), potassium acetate (CH₃CO₂K), ferrous sulfate heptahydrate (FeSO₄·7H₂O), and 3-(2-pyridyl)-3,0-diphenyl-1,2,4-triazine-4-4′-disulfonic acid sodium salt (C₂₀H₁₃N₄NaO₆S₂) were purchased from Sigma Chemical Company, USA. Dulbecco's modified Eagle medium (DMEM), heat-inactivated FBS, non-essential amino acids Solution (100x), and 0.25% trypsin-EDTA were purchased from Gibco, USA. Hydrogen peroxide and non-essential amino acid were purchased from Merck, USA. The scratch wound healing images were taken under an inverted microscope (Olympus, Japan), and the DCFH-DA assay was taken under a confocal fluorescent microscope (Olympus, Japan). The absorbance and fluorescence were evaluated with a microplate reader (BioTek Instruments, USA).

FISH was performed in an adult rat coronal brain section in vivo and rat NSCs in vitro using a FISH kit purchased from RiboBio (Guangdong, China) following the manufacturer’s protocol (Additional file 2). After washing the cells twice with PBS, they were fixed in 4% paraformaldehyde for 30 min and incubated in PBS with 0.1% Triton X-100 for 15 min. The hybridization probe was preheated before use. Cells were stained on the second day with Hoechst stain (Sigma, China) after washing twice with 4× saline sodium citrate (SSC) at 42 °C, once with 2× SSC, once with 1× SSC, and once with 1× PBS. Samples were then visualized using a confocal fluorescent microscope (Olympus, Japan). All experiments were performed in triplicate.

The 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCFDA) assay kit (Sigma-Aldrich, St Saint Louis, MO, USA) was used to evaluate intracellular reactive oxygen species upon UVB exposure [48 (link)]. The purpose of this experiment was to compare the antioxidant effects of the extract and celecoxib. Cells were seeded into an 8-chamber cover glass and treated with 20 µM of H₂DCFDA for 30 min in the dark. The cells were then washed with PBS and pretreated with the extract or celecoxib for 2 h before UVB exposure and incubated for a further 45 min. The fluorescent images were promptly taken using the confocal fluorescent microscope (Olympus, Japan). The ROS fluorescent intensity was calculated from the data average of three different areas by using Olympus software.

HEK293T/17 cells were grown on cover slips in 24 well tissue culture plates until 60% confluence. Then the cells were infected with DENV 2 at moi of 1. At 2 d.p.i, the cell culture media was removed and cells were washed with 1XPBS. The cells were fixed with 100% ice-cold methanol for 10–15 min at room temperature and allowed to dry for 15 min. Next, the cells were washed twice with 1XPBS/IFA for 5 min and permeabilized with 0.3% Triton-X 100 in 1X PBS/IFA for 10 min followed by washing twice with 0.03% Triton-X 100 for 5 min. The cells were blocked with 5% BSA in 1X PBS/IFA for 1 hr. After that, the cells were incubated with an appropriate primary antibody (Supplemental Table S4) at 4 °C for overnight. After washing, the cells were incubated with an appropriate secondary antibody (Supplemental Table S4), and a 1:200 dilution of 4′ 6-diamidino-2-phenyllindole (DAPI) at room temperature for 1 hr in the dark. After that the cells were washed six times with 0.03% Triton-X 100 for 5 min followed by mounting the cover slips onto glass slides using Prolong® Gold antifade reagent. The signals were observed under Olympus fluorescent confocal microscope.

The coding region of CaSBP11 without its termination codon was amplified and cloned into a pVBG2307: GFP vector (which contains a CaMV35S promoter that comes from PBI121 [63 (link)] and a GFP gene) between the BamHI and SmaI restriction sites to yield the final pVBG2307:CaSBP11:GFP plasmid (Supplementary Materials, Table S1) The recombinant fusion pVBG2307:CaSBP11:GFP plasmid was confirmed by sequencing performed by Sangon-Biotech Company (Shanghai, China). Then, the recombined vector (pVBG2307:CaSBP11:GFP) was transformed into the Agrobacterium tumefaciens strain GV3101 via the freeze-thaw method. Next, GV3101 cells carrying the pVBG2307:CaSBP11:GFP (CaMV35S:CaSBP11:GFP) and pVBG2307:GFP (CaMV35S:GFP) constructs were cultured overnight in Luria-Bertani (LB) medium with the appropriate antibiotics. Then, a prepared buffer (10 mM MES, pH 5.7, 10 mM MgCl2 and 200 μm acetosyringone) was used to create a cell suspension. The cell suspension (OD = 0.8) was then injected into the leaves of N. benthamiana with a needless syringe [64 (link)]. After injection, the plants were first cultured in darkness at 22 °C for 12 h and then cultured at 22/18 °C (day/night temperature) with a 16-h photoperiod for two days. After that, a fluorescent confocal microscope (Olympus, Tokyo, Japan) with a 488 nm excitation wavelength was used to detect green fluorescence.

The spinal cord tissues of the mice and BV2 cells with different treatments were collected. The BV2 cells were fixed with 4% paraformaldehyde and then blocked with 5% bovine serum albumin at room temperature for about 1 h. After that, the cells were incubated overnight with anti‐Iba1 (ab178846, 1:500, Abcam) and anti‐iNOS (ab178945, 1:500, Abcam) at 4°C. The cells were further incubated with the secondary antibodies and were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) for approximately 15 min. A fluorescent confocal microscope (Olympus, Japan) was used to assess all the images.
In addition, the spinal cord tissues were sliced into 5‐μm‐thick pieces and were further incubated overnight with anti‐Iba1 (ab178846, 1:500, Abcam) and anti‐iNOS (ab178945, 1:500, Abcam) at 4°C. The rest of the experimental operations were conducted in the same way that they were previously conducted.

The ORF of the CaSBP12 without a termination codon was cloned into a 35S::GFP vector with XbaI and KpnI restriction sites to yield the final plasmid 35S::CaSBP12::GFP. The CDS of CaSBP12 was amplified by PCR with XbaI and KpnI linker primers CaSBP12-GFP2-F and CaSBP12-GFP2-R (Supplementary Table S2). The recombinant fusion 35S:CaSBP12::GFP and 35S::GFP (used as the control) plasmids were introduced into onion epidermal cells by particle bombardment [42 (link)]. After transformation, tissues were incubated on MS agar medium under dark conditions at 28 °C for 24 h and then observed under a fluorescent confocal microscope (Olympus, Tokyo, Japan).