sheep polyclonal anti brdu  (Biodesign International Inc)

Journal: Bioengineering

Article Title: Development of an Eye Irritation Test Method Using an In-House Fabrication of a Reconstructed Human Cornea-like Epithelium Model for Eye Hazard Identification

doi: 10.3390/bioengineering11040302

Figure Lengend Snippet: ( a ) Monitoring the recovery of the iHCE-NY1 model using histological cross-sections. HE-stained tissue specimens of the iHCE-NY1 model’s incubation for 1- (post-incubation period (PIP) 1), 7-(PIP 7), 14-(PIP 14), and 21-days (PIP 21) post exposure to liquid chemicals are shown. At PIP 1, cells with enriched nuclei (yellow arrowheads) were observed, while viable cells (blue arrowheads) were observed from PIP 7. As the culture days increased to PIP 14 and PIP 21, more viable cells were observed. The gray dotted line indicates the position of the bottom of the iHCE-NY1 model. #6: γ-butyrolactone, #16: 2-methyl-1-pentanol, all bar indicates 50 µm. ( b ) Detection of respirating cells in the iHCE-NY1 model. Cells in S phase of the cell cycle that took up BrdU had cell nuclei revealed with red fluorescence. At post-incubation period (PIP) 1-day, few cells were in the S phase. From PIP 7-day onward, BrdU-positive S-phase cells were detected in all samples. #6: γ-butyrolactone, #16: 2-methyl-1-pentanol, all bar indicates 50 µm.

Article Snippet: Fixed and paraffin sections were prepared, and BrdU was detected using a sheep polyclonal anti-BrdU antibody (1:100, Capralogics, Hardwick, MA, USA) and an anti-sheep IgG antibody labeled with Alexa Fluor 594 (1:1000, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Staining, Incubation, Fluorescence

Journal: eLife

Article Title: CHARGE syndrome modeling using patient-iPSCs reveals defective migration of neural crest cells harboring CHD7 mutations

doi: 10.7554/eLife.21114

Figure Lengend Snippet: ( A ) xCELLigence. The migration index of iPSC-NCCs was measured using xCELLigence, by which migrating cells through microelectrode sensors were monitored automatically. ( B ) Representative curve of control (WD39; red) and CHARGE (CH2#1; blue) iPSC-NCCs migration index during 36 hr. Bar: SD. ( C ) Quantitative analysis of migration index after 20 hr of monitoring with xCELLigence, normalized to the control iPSC-NCCs migration index. Bar: SD. Biological replicates (the number of independent NCC inductions): control, N = 20 (WD39, N = 8; 201B7, N = 7; 1210B2, N = 5); CH1, N = 6 (CH1#7, N = 1, CH1#20, N = 1; CH1#25, N = 4); CH2, N = 11 (CH2#1, N = 5; CH2#16, N = 5; CH2#19, N = 1). Technical replicates: 2–4 per experiment. **p<0.01 (Turkey’s multiple comparisons test): The following file is available for , , . 10.7554/eLife.21114.017 Figure 5—source data 1. Raw data of xCelligence assay of iPSC-NCCs in vitro. Tabs 'Experiment 1' - 'Experiment 13': Raw data of migration indexes in each experiment. Each table in a sheet shows an independent experiment. Orange column shows migration index at 20 hr, and is used for quantitative analysis in . Tab 1: Statistical data of . Quantitative analysis of migration index after 20 hr of monitoring with xCELLigence, normalized to the control iPSC-NCCs migration index. Tab 2: Raw data and statistical data of . Control iPSC-NCCs migration index upon treatment with aphidicolin or vehicle for 36 hr. Tab 3: Raw data and statistical data of . BrdU assay of control and CHARGE iPSC-NCCs at 24 hr after replating. Tab 4: Raw data and statistical data of . Cell adhesion assay of control and CHARGE iPSC-NCCs to fibronectin.

Article Snippet: After 24 hr, the cells were fixed with PBS containing 4% PFA for 15 min at room temperature and immunostained with sheep polyclonal anti-BrdU antibody (1:500; Fitzgerald Industries International, Acton, MA).

Techniques: Migration, Control, In Vitro, BrdU Staining, Cell Adhesion Assay

Journal: eLife

Article Title: CHARGE syndrome modeling using patient-iPSCs reveals defective migration of neural crest cells harboring CHD7 mutations

doi: 10.7554/eLife.21114

Figure Lengend Snippet: ( A ) Representative curves of control iPSC-NCCs migration index upon treatment with aphidicolin (10 μg/ml) or vehicle (DMSO) for 36 hr. No significant difference was observed. (p=0.66; Two-way repeated measure ANOVA). Technical replicates: 4. Bar: SD. ( B ) BrdU assay of control and CHARGE iPSC-NCCs at 24 hr after replating. No significant difference between the control and CHARGE iPSC-NCCs was observed (p=0.86; Unpaired t test, Cohen’s d 0.12). Biological replicates (the number of independent NCC inductions): control, N = 5 (WD39, N = 3; 201B7, N = 2); CH1, N = 2 (CH1#25, N = 2); CH2, N = 2 (CH2#1, N = 2). Number of cells counted: control, N = 3244 (WD39, N = 2675; 201B7, N = 569); CH1 (CH1#25, N = 647); CH2 (CH2#1, N = 931). Graphs display the mean ± SD. ( C ) Cell adhesion assay of control and CHARGE iPSC-NCCs onto fibronectin. Each type of iPSC-NCC was cultured to semi-confluence in NC medium and was then replated (1 × 10 5 cells per ml) with WST-1 and incubated for 90 min. The absorbance of each well was measured and normalized to that of 201B7 iPSC-NCCs in each experiment. Bar: SD., Biological replicates: control (201B7, 1201C1), N = 6; CH1 (CH1#11, CH1#20), N = 3; CH2 (CH2#1, CH2#16), N = 10. Technical replicates: 4–6 per experiment., n.s., not significant (p=0.35 (Control vs CH1), p=0.99 (Control vs CH2); Dunnett’s multiple comparisons test).

Article Snippet: After 24 hr, the cells were fixed with PBS containing 4% PFA for 15 min at room temperature and immunostained with sheep polyclonal anti-BrdU antibody (1:500; Fitzgerald Industries International, Acton, MA).

Techniques: Control, Migration, BrdU Staining, Cell Adhesion Assay, Cell Culture, Incubation