Immunofluorescence localization assays were performed as described by Martínez-Macías et al. (47 (link)). First, seedling tissue samples were used for nuclei preparation. The nuclei preparations were incubated at room temperature with different combinations of anti-Flag (F7425, Sigma), anti-Flag (F1804, Sigma), anti-Myc (05-724, Millipore), H3K9me1 (07-352, Millipore), S9.6 (from Q. Sun’s laboratory, Tsinghua University), and anti-γH2AX (4418-APC-020, Trevigen) primary antibodies overnight, after which they were incubated with mouse Alexa594 (A23410, Abbkine)–conjugated or rabbit Alexa-488 (A23220, Abbkine)–conjugated secondary antibodies for 2 hours at 37°C. After washing with phosphate-buffered saline, DNA was counterstained using DAPI in Prolong Gold Antifade Mountant (Invitrogen). Nuclei were observed with a confocal microscope, Leica TCS SP8 STED 3× (Leica).
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Immunofluorescence Localization of Nuclear Proteins
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Alexa594
Antibodies
Confocal microscope
Dapi
Gold
Immunofluorescence assays
Mouse
Nuclei
Phosphate
Rabbit
Saline
Tissue
Corresponding Organization : Center for Life Sciences
Other organizations : China Agricultural University
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Variable analysis
independent variables
- Different combinations of primary antibodies (anti-Flag, anti-Myc, H3K9me1, S9.6, anti-γH2AX)
- Localization of proteins/histone marks in the nucleus
- Seedling tissue samples
- Nuclei preparation
- Incubation with primary antibodies overnight
- Incubation with secondary antibodies for 2 hours at 37°C
- Washing with phosphate-buffered saline
- DNA counterstaining with DAPI
- Observation with confocal microscope
- Positive control: Not specified
- Negative control: Not specified
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