Tissues were fixed in 4% paraformaldehyde in PBS overnight at 4°C
and incubated in 30% sucrose in PBS at 4°C overnight. Tissues were
embedded in tissue freezing medium and cut 8 μm thicknesses. For antigen
retrieval, either 1 mM EDTA with 0.05% Tween 20 in boiling water or epitope
retrieval solution (IHC World) in a steamer (IHC-Tek Epitope Retrieval Streamer
Set) were used, then sections were blocked with 10% serum from the host animal
of secondary antibodies, and incubated with primary anti-bodies overnight at 4
°C. The sections were subsequently washed with PBS and incubated with
corresponding secondary antibodies conjugated to Alexa Fluor 488 or 555
(Invitrogen). The slides were mounted in antifade mounting medium (Vector
Laboratories, Burlingame, California). Primary antibodies used are as follows:
anti-phospho histone H3 Ser10 (Millipore 06–570, 1:100), anti-aurora B
(Sigma A5102, 1:100), anti-troponin T, cardiac isoform Ab-1, clone 13–11
(Thermo Scientific MS-295-P1, 1:100), anti-sarcomeric α-actinin (Abcam,
ab68167, 1:100), anti-8 hydroxyguanosine (Abcam ab62623, 1:50),
anti-phosphorylated ATM (Santa Cruz Biotechnology sc-47739, 1:100),
Anti-phospho-Histone gamma H2AX (Millipore-Sigma 05–636). DAPI was used
for the nuclear staining.
and incubated in 30% sucrose in PBS at 4°C overnight. Tissues were
embedded in tissue freezing medium and cut 8 μm thicknesses. For antigen
retrieval, either 1 mM EDTA with 0.05% Tween 20 in boiling water or epitope
retrieval solution (IHC World) in a steamer (IHC-Tek Epitope Retrieval Streamer
Set) were used, then sections were blocked with 10% serum from the host animal
of secondary antibodies, and incubated with primary anti-bodies overnight at 4
°C. The sections were subsequently washed with PBS and incubated with
corresponding secondary antibodies conjugated to Alexa Fluor 488 or 555
(Invitrogen). The slides were mounted in antifade mounting medium (Vector
Laboratories, Burlingame, California). Primary antibodies used are as follows:
anti-phospho histone H3 Ser10 (Millipore 06–570, 1:100), anti-aurora B
(Sigma A5102, 1:100), anti-troponin T, cardiac isoform Ab-1, clone 13–11
(Thermo Scientific MS-295-P1, 1:100), anti-sarcomeric α-actinin (Abcam,
ab68167, 1:100), anti-8 hydroxyguanosine (Abcam ab62623, 1:50),
anti-phosphorylated ATM (Santa Cruz Biotechnology sc-47739, 1:100),
Anti-phospho-Histone gamma H2AX (Millipore-Sigma 05–636). DAPI was used
for the nuclear staining.