Immunofluorescence Protocol for Protein Localization

Immunofluorescence was carried out as described in Chang et al., 2014 (link). Briefly, cells were fixed and permeabilized with 100% methanol, blocked with 5% BSA in 1X PBS, and incubated with antibodies at 4°C overnight. Cells were mounted using ProLong Gold Antifade with DAPI (Life Technologies; P36935). Primary antibodies: Androgen Receptor (AR N-20) (Santa Cruz; sc-816), p62/SQSTM1 (Abnova; H00008878-M01). Fluorescently labeled secondary antibodies: Alexafluor 488, goat anti-mouse (Invitrogen; A11001), Alexafluor 568, goat anti-rabbit (Invitrogen; A11061).

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Chang M., Patel V., Gwede M., Morgado M., Tomasevich K., Fong E., Farach-Carson M, & Delk N. (2014). IL-1β induces p62/SQSTM1 and represses androgen receptor expression in prostate cancer cells. Journal of cellular biochemistry, 115(12), 2188-2197.

Publication 2014

ProLong Gold Antifade with DAPI Androgen Receptor (AR N-20) p62/SQSTM1 Alexafluor 488, goat anti-mouse Alexafluor 568, goat anti-rabbit

Androgen receptor Antibodies Cells Dapi Goat Gold Immunofluorescence Methanol Mouse Rabbit

Corresponding Organization : The University of Texas at Dallas

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Variable analysis

independent variables

Primary antibodies: Androgen Receptor (AR N-20) (Santa Cruz; sc-816), p62/SQSTM1 (Abnova; H00008878-M01)
Fluorescently labeled secondary antibodies: Alexafluor 488, goat anti-mouse (Invitrogen; A11001), Alexafluor 568, goat anti-rabbit (Invitrogen; A11061)

dependent variables

Immunofluorescence staining patterns

control variables

Cells were fixed and permeabilized with 100% methanol
Cells were blocked with 5% BSA in 1X PBS
Cells were incubated with antibodies at 4°C overnight
Cells were mounted using ProLong Gold Antifade with DAPI (Life Technologies; P36935)

controls

No positive or negative controls were explicitly mentioned.

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