Mouse anti brdu antibody

MCF-7 cells were seeded on 6-well plate and were grown overnight before transfection. Fresh medium containing 10 μmol/L BrdU (Sigma, USA) was used to incubate all groups for 4 hours prior to immunofluorescence staining with mouse anti-BrdU antibody. After fixation with 4% paraformaldehyde in PBS, the cells were incubated overnight with a mouse anti-BrdU antibody (NeoMarkers, USA) and then treated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Dako, Glostrup, Denmark). Propidium iodine (PI) (Sigma) (50 μg/mL) was used to stain nuclei as the control to all cells in each group. The labeling index was expressed as the number of positively labeled nuclei/total number of nuclei.

HUVECs were allowed to reach 50–70% confluency and then incubated with BrdU (25 μM) (Sigma-Aldrich) at 37°C for 2 hours. Cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with PBS containing 0.3% Triton X-100 for 15 minutes at room temperature, and incubated with 2 M HCl for 30 minutes at 37°C. After DNA denaturation, the remaining HCl was neutralized by incubating cells with 0.1 M borate buffer. Cells were then washed three times with PBS containing 0.05% Tween 20 (PBST), blocked with 2% goat serum in PBST for 30 minutes at 37°C, followed by incubation with a mouse anti-BrdU antibody (1 : 1000, Sigma-Aldrich) for 30 minutes at 37°C. After washing three times with PBST, cells were incubated with Alexa Fluor 488 goat anti-mouse (1 : 400, Invitrogen) for 30 minutes at 37°C and counterstained with DAPI. Cells were imaged using a fluorescence microscope at a magnification of 100x. BrdU positive cells were counted and normalized to the total cells in the same field.

At 12 days after ischemia, the rats were deeply anesthetized with sodium pentobarbital (100 mg/kg, IP) and then transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3). After transcranial perfusion, the animals' brains were removed and postfixed in 4% paraformaldehyde for one week. Next, we processed the tissues and a series of adjacent 5 μm thick sections were cut from paraffin blocks in the coronal plane. Immunohistochemical staining was used to detect the distribution of transplanted BMSCs in the hippocampus, as follows: deparaffinization of samples in xylol (Merck, Germany), hydration in a graded alcohol series (100, 96, 80, and 70%), incubation in 50% formamide (Merck, Germany), incubation in 2x standard sodium citrate (SSC, Merck, Germany) for 2 h at 65°C, incubation in 2 N HCl (Merck, Germany) for 30 min at 37°C, rinse in 0.1 N boric acid (Merck, Germany, pH 8.5) for 10 min, followed by a PBS wash, incubation with mouse anti-Brdu antibody (Sigma, Germany) overnight at 4°C, rinse in PBS (3 times for 10 min), incubation with secondary antibody conjugated with horse radish peroxidase (goat anti-mouse IgG) (Sigma, Germany) for 2 h, and incubation with diaminobenzidine tetrachloride hydrate (DAB, Sigma, Germany) for 5 min, after which the slides were counterstained with hematoxylin, mounted, and inspected under a light microscope.

Cells were plated onto polyornithine (50 μg/ml) coated 96-well plates at a density of 1×10⁴ cells. Cells were treated with either DMSO or curcumin at two time points (48 and 72 h). Adherent cells and neurospheres were then fixed in 4 % paraformaldehyde for 30 min. The cells were washed with 0.2 % Triton-X 100 in 0.1 M PBS (pH 7.4) three times for 5 min. DNA was denatured by exposing the cells to acid (2 M HCl, 30 min at 37 °C). After that, borate buffer (0.1 M) was added to the cells for 12 min at room temperature. Then, 5 % normal goat serum in 0.1 M PBS (pH 7.4) was added to block endogenous peroxidases. Next, the cells were incubated overnight with mouse anti-BrdU antibody (1:1000, Sigma, Germany) and thereafter exposed to FITC conjugate anti-mouse IgG (1:1000, Sigma, Germany) for BMSCs or Alexa-Fluor 647-conjugated anti-mouse IgG (1:500, Abcam USA) for NS/PCs for 2 h at room temperature. Each experiment was repeated three times.
Proliferating cells and neurospheres were incubated by 10 μM/l BrdU for 48 h and analyzed by immuno-staining [18 (link)]. Fluorescent density of Brdu positive cells were measured using image J software [19 (link), 20 (link)]. The study had the endorsement of the ethical committee of Tehran University of Medical Sciences.

The various experimental tissue sections of E. eugeniae were subjected to IHC by the protocol of published paper [40 (link)]. 6 μm sections were subjected to immunohistochemistry to observe the TCTP expression. After the paraffin was removed from the sections, they were treated with 10% H₂O₂ (Cat.1072090500; Merck, India) and 10% methanol (Cat. AS059; Himedia, India) in 1X PBS to block the endogenous peroxidise activity, followed by trypsin treatment (0.1% trypsin in 0.1% CaCl₂ for 10 minutes). The non-specific blocking was carried out by keeping the tissue slides in 2% BSA for 1 hour at room temperature. The IHC was performed individually by primary antibodies of anti-TCTP antibody, anti-Phospho H3 [pSer¹⁰] antibody (Cat. H0412, Sigma-Aldrich, India) and mouse anti-BrdU antibody (Cat. B8434, Sigma-Aldrich, India) at a concentration of 1:100, 1:200, 1:100, respectively, in 2% BSA, overnight at 4°C. After the PBS wash had been rendered, sections were incubated with secondary antibody Goat anti-mouse IgG conjugated with horseradish peroxidise (Cat.031050, Sigma-Aldrich, India) at a dilution of 1:2000 in 1X PBST. Diaminobenzidine (Cat.E733; Amresco, USA) was used as a developer and sections were counter-stained with Mayer’s hematoxylin (Cat. MHS1, Sigma-Aldrich). The slides were mounted with DPX and documented under an Olympus BX53 microscope.

BrdU, mouse anti-BrdU antibody, PI, L-161,982 and celecoxib were purchased from Sigma-Aldrich. 16,16-dimethyl-PGE₂, XAV939, AH6809 and forskolin were from Tocris Bioscience (Bristol, UK). Niflumic acid was from Cayman Chemical (Ann Arbor, MI, USA). All other chemicals were of analytical grade.