Ab8980

Cells were lysed in lysis buffer containing protease inhibitor. Protein concentration was determined by the BCA protein assay kit (Novagen, Merck Group, Madison, USA). Equal amounts of denatured proteins were separated by SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 5% nonfat milk at room temperature for 1 h, and then incubated with primary antibody, followed by horseradish peroxidase-conjugated secondary antibody. Protein expression levels were detected using Image Lab software (Bio-Rad, CA, USA). Antibodies against lamin-A (mouse, ab-8980) and γ-H2AX (rabbit, ab81299) were from Abcam, U.K. Antibodies against Rad50 (rabbit, 3427) and Ku80 (rabbit, 2180) were from Cell Signaling Technology, USA. Antibody against Flag (mouse, F1804) was from sigma, USA. Antibodies against His (mouse, 66005–1-Ig) and Hsp90 (rabbit, 13171-1-AP) were from Proteintech, China.

Specific antibodies recognizing human H1 variants used for ChIP/ChIP-seq were: anti-H1.0/H5 clone 3H9 (Millipore, 05–629-I), anti-H1.2 (Abcam, ab4086), anti-H1.4 (Invitrogen, 702876), anti-H1.5 (Invitrogen, 711912) and anti-H1X (Abcam, ab31972). ChIP-seq of H3K9me3 was performed using anti-H3K9me3 (Abcam, ab8898). Other antibodies used were: anti-H1.0 (Abcam, ab11079), anti-H1.3 (Abcam, ab24174), anti-H1.5 (Abcam, ab24175), anti-H3 (Abcam, ab1791) and anti-Lamin A (Abcam, ab8980).

Fibers were PFA-fixed and Triton-permeabilized as described above (Nuclear organization of single fibers). Fibers were treated with mouse-on-mouse block (Vector Laboratories, BMK-2202) for 1 hour, blocked in 10% goat serum in PBS (Sigma-Aldrich, G9023) for 30 minutes, and treated with primary antibodies diluted in goat serum blocking buffer. Prelamin A was visualized using as primary antibody Santa Cruz Biotechnology Inc. sc-6214, c-20 (secondary antibody: Alexa Fluor 488, Invitrogen, A-11078) and Pax7 using Developmental Studies Hybridoma Bank (DSHB) AB 528428 as primary antibody (secondary antibody: Alexa Fluor 488, Invitrogen, A-11001). Lamin A (Abcam, ab8980) and nesprin-1 (Abcam, ab192234) were matched with Alexa Fluor 488 and 594, respectively (Invitrogen, A-11001 and A-11012). Acetyl-histone H3 (Lys9/Lys14) antibody (Cell Signaling Technology, 9677) matched with Alexa Fluor 594, respectively (Invitrogen, A-11012).

The following primary antibodies were used for immunofluorescence (IF) and/or western blotting (WB): mouse anti-lamin A [ab8980, Abcam, Paris, France (IF: 1/100, WB: 1/200)]; rabbit anti-lamin C [BP4505, Acris, Herford, Germany (IF: 1/100, WB: 1/230)]; mouse anti-lamin A/C [MAB3211, Merck Millipore, Massachusetts, United States (WB: 1/200)]; goat anti-lamin A/C [sc6215, Santa Cruz, Texas, United States (WB: 1/200)]; rabbit anti-lamin A/C [sc20681, Santa Cruz (WB: 1/200)]; rabbit anti-lamin B1 [ab16048, Abcam (WB: 1/250)]; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [MAB374, Merck Millipore (WB: 1/40,000)].
For IF, the following isotype antibodies were used as negative controls: mouse IgG (015-000-003, Jackson ImmunoResearch, Suffolk, UK); and rabbit IgG (AB-105-C, R&D, Minnesota, United States).

Eyes were fixed in 4% paraformaldehyde for 1 h before cryoprotecting in 20% sucrose and embedding in OCT (RA Lamb) and freezing in isopentane precooled in liquid nitrogen. Cryosections were cut at 18-μm thick and all sections were collected for analysis. For histology and cell counting, cryosections were air-dried for 30 min and washed in PBS containing Hoechst 33342 (10 μM) for 5 min before mounting. For immunohistochemistry, sections were blocked in 5% goat serum and 1% bovine serum albumin and triton X-100 (0.05% in PBS at RT for 1 h. Primary antibody was incubated overnight at 4 °C. After washing, sections were incubated with secondary antibody for 2 h at RT, washed and counter-stained with Hoechst 33342 (10 μM). Primary antibodies used included: LaminB antibody (mouse monoclonal; Abcam Ab8980; 1:200); rod α-transducin (rabbit polyclonal; Santa Cruz SC389; 1:1,000); CRE Recombinase (Mouse monoclonal; Millipore MAB3120; 1:200). Alexa fluor 488, 546 and 633 secondary antibodies (Invitrogen-Molecular Probes) were used at a 1:500 dilution. Negative controls omitted the primary antibody. Retinae receiving rEGFP were examined both unstained, as is typical for assessing retinae receiving retinal transplants9 (link)12 (link) and following immunostaining with a directly conjugated chicken anti-GFP antibody (Invitrogen-Molecular Probes; 1:200 for 2 h before mounting).