Immunofluorescence Imaging of PDGF and PDGFR in 3D Cell Cultures

IHCs were performed on paraffin sections (4–5 μm) of NMF and BJ3Z cells cultured in 3D colonies as described [30 (link)]. They used antibodies directed against PDGF-A (sc-7958; rabbit polyclonal, Santa Cruz), PDGF-B (sc-7878; rabbit polyclonal, Santa Cruz), PDGFR-α (AF1062; goat polyclonal, R&D systems) and PDGFR-β (AF1042; goat polyclonal, R&D systems). Briefly, sections were deparaffinized and antigen retrieval was performed in a pressure cooker (Bio-care Medical) at 20 psi for 5 min in citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0). Sections were blocked 30 min with 10% normal goat serum and primary antibodies were applied for 1 hr. Fluorescent secondary antibodies were: Alexa fluor 555 (red) donkey anti-goat IgG (1:300) and Alexa fluor 488 (green) goat anti- rabbit IgG (1:400; both Invitrogen). Cell nuclei were counterstained with 4-6-Diamidino-2-phenylindole (DAPI). Fluorescent images were obtained using a Nikon Eclipse E600 fluorescent microscope coupled to a RGB-MSC micro color camera, and Image Pro Plus software version 4.5 (Media Cybernetics, Silver Spring, MD).

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Pinto M.P., Dye W.W., Jacobsen B.M, & Horwitz K.B. (2014). Malignant stroma increases luminal breast cancer cell proliferation and angiogenesis through platelet-derived growth factor signaling. BMC Cancer, 14, 735.

Publication 2014

AF1042 pressure cooker Alexa fluor 488 (green) goat anti- rabbit IgG Image Pro Plus software version 4.5

Alexa fluor 488 Alexa fluor 555 Anti igg Antibodies Antigen Buffer Cell nuclei Cells Citrate Donkey E600 Fluorescent antibodies Goat Microscope Paraffin Pdgf Pdgf a Pdgfr β Pressure Rabbit Serum Silver Sodium citrate Tween 20

Corresponding Organization : Universidad Andrés Bello

Other organizations : University of Colorado Anschutz Medical Campus

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Variable analysis

independent variables

Cell type (NMF and BJ3Z cells)

dependent variables

Expression of PDGF-A, PDGF-B, PDGFR-α, and PDGFR-β

control variables

Paraffin sections (4–5 μm) of cells cultured in 3D colonies
Antigen retrieval in a pressure cooker (20 psi for 5 min in citrate buffer)
Blocking with 10% normal goat serum
Primary antibody incubation (1 hr)
Fluorescent secondary antibodies (Alexa fluor 555 donkey anti-goat IgG and Alexa fluor 488 goat anti-rabbit IgG)
DAPI counterstaining of cell nuclei
Fluorescent imaging using a Nikon Eclipse E600 microscope

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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