Immunofluorescent Staining of Cells

The immunofluorescent staining of cells grown on glass slides was performed as described elsewhere [7 (link), 8 (link), 20 (link)]. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and incubated at 4°C overnight with rabbit polyclonal antibodies against TIN2 [20 (link)], γ-H2AX (i.e., phosphorylated-H2AX) (Upstate), or AR (AR-N20; Santa Cruz), or mouse monoclonal antibodies against AR (AR-414; Santa Cruz), AR-V7 (AG10008; Precision) or pATM (10H11-E12, which detects phosphorylation of ATM at serine 1981; Cell Signaling). Cells were then washed and stained with FITC-labeled goat-anti-rabbit-IgG and/or Texas Red-labeled goat-anti-mouse-IgG (Molecular probes) secondary antibodies [7 (link), 8 (link)]. Images of cells were acquired on an LSM-410 confocal microscope (Zeiss). Labeled foci were counted in enlarged photographs.

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Reddy V., Iskander A., Hwang C., Divine G., Menon M., Barrack E.R., Reddy G.P, & Kim S.H. (2019). Castration-resistant prostate cancer: Androgen receptor inactivation induces telomere DNA damage, and damage response inhibition leads to cell death. PLoS ONE, 14(5), e0211090.

Publication 2019

AR-N20 FITC-labeled goat-anti-rabbit-IgG LSM-410 confocal microscope

Anti igg Antibodies Cells Confocal microscope Fitc Goat Immunofluorescent Molecular probes Monoclonal antibodies Mouse Paraformaldehyde Phosphorylation Rabbit Serine Triton x 100

Corresponding Organization : Henry Ford Hospital

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Variable analysis

independent variables

Antibody type (rabbit polyclonal or mouse monoclonal)
Antibody target (TIN2, γ-H2AX, AR, AR-V7, pATM)

dependent variables

Fluorescence intensity of labeled foci
Number of labeled foci per cell

control variables

Cell growth conditions (cells grown on glass slides)
Cell fixation (4% paraformaldehyde)
Cell permeabilization (0.5% Triton X-100)
Incubation conditions (4°C overnight)
Imaging method (confocal microscopy)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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