Immunofluorescence Staining of Stem Cells

Immunofluorescence was performed on cells grown directly on slides as previously described (Chadwick and Willard 2002 (link)). Except, the maximum slide seed area was delimited using a Super PAP Pen (IM3580, Beckman Coulter^®) and coated with 700µl of Matrigel™ or 0.1% Gelatin prior to seeding hESCs or EBOGs, respectively. Additionally, hESCs were allowed to adhere for 1hr in complete StemPro^® media supplemented with ROCK Inhibitor Y-27632 (ROCKi, SCM075, Millipore™), the media was then replaced with fresh complete StemPro^® media (-ROCKi), and allowed to recover overnight. Antibodies used for indirect Immunofluorescence included rabbit anti-H3K4me2 (07–030, EMD Millipore), mouse anti-H3K27me3 (ab6002, abcam), rabbit anti-NANOG (3580S, Cell Signaling Technology), mouse anti-SSEA4 (4755S, Cell Signaling Technology), goat anti-SOX2 (AF2018, R&D Systems), and rabbit anti-OCT4 (2750S, Cell Signaling Technology). Conjugate secondary antibodies (Alexa-Fluor^®) were obtained from Life Technologies Corporation. DNA was counterstained using the VECTASHIELD^® mounting medium with DAPI from VECTOR Laboratories. Imaging was performed on an Olympus IX71 operated by the DeltaVision pDV, deconvolved with softWoRx 5.5.1 (DeltaVision), and compiled using Adobe Photoshop CS6 (Adobe Systems).

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Figueroa D.M., Darrow E.M, & Chadwick B.P. (2015). Two novel DXZ4-associated long noncoding RNAs show developmental changes in expression coincident with heterochromatin formation at the human (Homo sapiens) macrosatellite repeat. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology, 23(4), 733-752.

Publication 2015

rabbit anti-H3K4me2 mouse anti-H3K27me3 ( rabbit anti-NANOG mouse anti-SSEA4 goat anti-SOX2 (AF2018 rabbit anti-OCT4 VECTASHIELD® mounting medium

Antibodies Cells Dapi Gelatin Goat Hescs Immunofluorescence Indirect immunofluorescence Matrigel Mouse Oct4 Rabbit Sox2 Ssea4 Vector Y 27632

Corresponding Organization :

Other organizations : Florida State University, National Institutes of Health, National Heart Lung and Blood Institute

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Variable analysis

independent variables

Cell type (hESCs or EBOGs)
Coating material (Matrigel or 0.1% Gelatin)
ROCK Inhibitor Y-27632 treatment (presence or absence)

dependent variables

Immunofluorescence staining patterns for H3K4me2, H3K27me3, NANOG, SSEA4, SOX2, and OCT4

control variables

Cells grown directly on slides
Maximum slide seed area delimited using a Super PAP Pen
Complete StemPro® media for cell culture
Primary and secondary antibodies used for indirect Immunofluorescence
DNA counterstained using VECTASHIELD® mounting medium with DAPI
Imaging performed on an Olympus IX71 microscope and processed using DeltaVision and Adobe Photoshop software

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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