rat anti brdu primary antibody  (Bio-Rad)

Journal: bioRxiv

Article Title: A Nerve-Fibroblast Axis in Mammalian Lung Fibrosis

doi: 10.1101/2024.09.09.611003

Figure Lengend Snippet: (A-D) Immunofluorescence imaging displayed BrdU (green) and nuclear staining with propidium iodide (PI) (red) in MRC5 human lung fibroblasts. A dose- dependent increase in BrdU incorporation was observed in MRC5 cells stimulated with increasing concentrations of noradrenaline (NA), peaking at 25 µM (D, P = 0.0159). This response was reversed upon co-incubation with terazosin (E, P = 0.0441). (F) MTT assays demonstrated a significant reduction in the number of viable cells in MRC5 human lung fibroblasts and IPF fibroblasts when stimulated with NA and treated with terazosin ( P = 0.0493 and P = 0.0156, respectively). This effect was not observed in normal human lung fibroblasts, indicating that IPF fibroblasts are poised to receive and respond to noradrenergic signals via an α1-AR dependent mechanism. (G-I) Immunofluorescence imaging demonstrated expression of ADRA1D (red) and α-SMA (green), with nuclear staining by DAPI (blue) in human precision-cut lung slices. Following exposure to a fibrotic cocktail, a marked increase in α-SMA (ACTA2) expression was observed in stromal cells adjacent to alveoli, with some cells showing co- expression of ADRA1D (white arrows, G, H). This expression pattern was consistent with trichrome staining and matched ACTA2 expression quantified by PCR analysis (J, P = 0.0236). The introduction of terazosin to the culture media reversed these fibrotic effects (I, J, P = 0.0106). Images were captured at 20x magnification. Data are presented as mean ± SEM or median ± IQR, with statistical analysis performed using Student’s t-test for normally distributed data and Mann-Whitney or Kruskal-Wallis tests with Dunn’s multiple comparisons for non- normally distributed data. * P < 0.05, ** P < 0.01. ADRA1D, α1-adrenoreceptor subtype D; α-SMA, alpha-smooth muscle actin; BrdU, bromodeoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; FC, fibrotic cocktail; IPF, idiopathic pulmonary fibrosis; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; NA, noradrenaline; NHLF, normal human lung fibroblast; PCR, polymerase chain reaction; PI, propidium iodide; TZ, terazosin.

Article Snippet: After this incubation, the cells were stained with rat anti-BrdU primary antibody (1:500, BioRad, MCA2483T) for one hour at room temperature.

Techniques: Immunofluorescence, Imaging, Staining, BrdU Incorporation Assay, Incubation, Expressing, MANN-WHITNEY, Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: A New Method for In Vitro Detection of Bromodeoxyuridine in Serum: A Proof of Concept in a Songbird Species, the Canary

doi: 10.1371/journal.pone.0063692

Figure Lengend Snippet: (A–F) Cultured cells stained for BrdU, after incubation with BrdU at different concentrations (as indicated at the bottom of each image). Images were consistently modified to enhance contrast and remove colour, and are all shown to the same scale. (A) At very low concentrations, very few cells were labelled even weakly. (B–F) As BrdU concentration increased, the proportion of visible cells that were weakly-labelled (w), moderately-labelled (m), or strongly-labelled (s) increased. Every culture plate also contained some unlabelled cells (arrowhead). (G) Total numbers of labelled cells (weakly+moderately+strongly) reached a plateau with BrdU concentrations above 50 µg/mL; note the break in the x-axis scale . At lower concentrations, however, (inset) there was a strong linear relationship between BrdU concentration and the total number of labelled cells (Y = 1.651 X, line forced through [0,0]; confidence interval for slope = 1.403 to 1.898, r = 0.9606, p<0.0001). Dashed lines show 95% confidence interval around the (solid) line of best fit.

Article Snippet: Cells were then incubated with 0.6% hydrogen peroxide in PBS, rinsed, incubated 10 min in 2N HCl followed by 5 min in 0.1 M sodium borate buffer (pH 8.5), rinsed again, and blocked 8 min in 10% normal horse serum in PBS with 0.02% Triton-X100 (PBS-T) followed by incubation for 2 h at 4°C with monoclonal rat anti-BrdU primary antibody (AbD Serotec, 1∶1000) in PBS-T. Plates were rinsed three times in PBS, followed by incubation for 30 min at room temperature in donkey anti-rat biotinylated secondary antibodies (Jackson Immunoresearch, 1∶2000) in PBS-T. After another three rinses in PBS, plates were incubated 30 min at room temperature with an avidin-biotin complex (Vectastain Elite kit, Vector Laboratories).

Techniques: Cell Culture, Staining, Incubation, Modification, Concentration Assay

Journal: PLoS ONE

Article Title: A New Method for In Vitro Detection of Bromodeoxyuridine in Serum: A Proof of Concept in a Songbird Species, the Canary

doi: 10.1371/journal.pone.0063692

Figure Lengend Snippet: (A). Percentage (means ± SD) of the total numbers of cells visible in culture that were immunoreactive for BrdU after incubation with serum collected from adult canaries at various times after BrdU injection. All individual male (black spots) and female (open circles) data points are represented in the figure. (B) Average concentrations (means ± SD) of BrdU in these canary samples based on the calibration curve illustrated in the inset of . (C–D) Concentrations of BrdU in blood samples collected in two female Japanese quail (C) or one male mouse (D) at various times after a single BrdU injection.

Article Snippet: Cells were then incubated with 0.6% hydrogen peroxide in PBS, rinsed, incubated 10 min in 2N HCl followed by 5 min in 0.1 M sodium borate buffer (pH 8.5), rinsed again, and blocked 8 min in 10% normal horse serum in PBS with 0.02% Triton-X100 (PBS-T) followed by incubation for 2 h at 4°C with monoclonal rat anti-BrdU primary antibody (AbD Serotec, 1∶1000) in PBS-T. Plates were rinsed three times in PBS, followed by incubation for 30 min at room temperature in donkey anti-rat biotinylated secondary antibodies (Jackson Immunoresearch, 1∶2000) in PBS-T. After another three rinses in PBS, plates were incubated 30 min at room temperature with an avidin-biotin complex (Vectastain Elite kit, Vector Laboratories).

Techniques: Incubation, Injection