"In brief, H1 cells were grown to 70% confluency, the medium was removed and cells were fixed in 4% and 8% paraformaldehyde in 250 mM HEPES-NaOH (pH 7.6; 10 min and 2 h, respectively), gently scrapped and softly pelleted, before embedding (>2 h) in saturated 2.1 M sucrose in PBS and frozen in liquid nitrogen on copper sample holders. Frozen samples were stored in liquid nitrogen."
"For laser microdissection, cryosections on PEN membranes were washed, permeabilized and incubated (2 h, room temperature) in blocking solution (1% BSA (w/v), 5% FBS (w/v, Gibco 10270), 0.05% Triton X-100 (v/v) in PBS). After incubation (overnight, 4 °C) with primary anti-pan-histone (1:50) antibody (Merck, MAB3422) in blocking solution, the cryosections were washed three to five times for 30 min in 0.025% Triton X-100 in PBS (v/v) and immunolabelled (1 h, room temperature) with secondary antibodies in blocking solution, followed by three (15 min) washes in PBS."
H1 cells were grown to 70% confluency, fixed sequentially in 4% (10 min) and 8% (2 h) paraformaldehyde in 250 mM HEPES-NaOH (pH 7.6), scraped, pelleted, embedded in saturated 2.1 M sucrose in PBS (>2 h) and frozen in liquid nitrogen on copper holders for storage. Ultrathin cryosections (~220 nm) were prepared with a Leica ultracryomicrotome, captured on sucrose-PBS drops and transferred to 4 µm PEN steel frame slides for laser microdissection. Sucrose was removed with filtered PBS and ultrapure water washes. Cryosections were permeabilized, blocked, incubated overnight with anti-pan-histone primary antibody (1:50) at 4°C, washed, immunolabeled with secondary antibodies, and washed again. Nuclear staining was visualized and individual nuclear profiles were laser microdissected and collected in PCR adhesive caps in multiplexGAM mode, pooling three NPs per cap.
Read at Nature
Unable to calculate read time
Collection
[
|
...
]