Immunofluorescence Analysis of Spheroid β-Catenin

HT29-s (~8,000 spheroids) were grown on poly-L-lysine (0.01%) coated coverslips. Immunofluorescence was performed in treated and untreated spheroids as described^{13 (link)}. Anti-phospho/total-β-catenin (1:30) antibody were used for overnight incubation at 4°C. Alexa Flour 488 labeled goat anti-mouse IgG (H+L) (1:300) (Invitrogen) and DAPI were used. Keyence Fluorescence microscopy was used to take the images with 200X magnification.

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Sokolov D., Sharda N., Giri B., Hassan M.S., Singh D., Tarasiewicz A., Lohr C., von Holzen U., Kristian T., Waddell J., Reiter R.J., Ahmed H, & Banerjee A. (2022). Melatonin and Andrographolide synergize to inhibit the colospheroid phenotype by targeting Wnt/beta-catenin signaling. Journal of pineal research, 73(1), e12808.

Publication 2022

Alexa Flour 488 labeled goat anti-mouse IgG (H+L)

Anti igg Antibody Dapi Flour Fluorescence microscopy Goat Ht29 Immunofluorescence Lysine Mouse Poly β catenin

Corresponding Organization : University of Maryland, Baltimore

Other organizations : Maryland Department of Health, Indiana University, University of Basel, VA Maryland Health Care System, University of Mary, Star Center, The University of Texas Health Science Center at San Antonio, The University of Texas Health Science Center at Houston

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Variable analysis

independent variables

Treatment condition (treated vs. untreated spheroids)

dependent variables

Phosphorylation and total levels of β-catenin

control variables

HT29-s cell line (~8,000 spheroids)
Poly-L-lysine (0.01%) coated coverslips
Immunofluorescence protocol as described in reference 13
Anti-phospho/total-β-catenin (1:30) antibody
Overnight incubation at 4°C
Alexa Flour 488 labeled goat anti-mouse IgG (H+L) (1:300) (Invitrogen)
DAPI staining
Keyence Fluorescence microscopy with 200X magnification

controls

Untreated spheroids (negative control)

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