A21441

The spermatocyte spreads and seminiferous tubules squashes were blocked for 30 min with 1% BSA and 0.05% Tween in PBS (PBS-T) and then were incubated overnight at 4 °C with the following primary antibodies: Sycp3 (Abcam, ab15093 and ab97672, 1:800; and Santa Cruz, sc-20845, 1:20); Sycp1 (Abcam, ab15087, 1:800) Dmc1 (Santa Cruz, sc-22768, 1:50); Trf1 (Alpha Diagnostics, TRF12-S, 1:50); Sun1 (Abcam, ab103021, 1:50); H1t (gift from Dr Mary Ann Handel, The Jackson Laboratory, Bar Harbor, Maine, USA, 1:200) H3K9-3me (Milipore, 05-1250, 1:2000); γH2AX (Milipore, 05-636, 1:300); Mlh1 (Becton Dickinson, 551091, 1:100); Rad51 (Santa Cruz, sc-8349, 1:100) Terb-1 (affinity-purified rabbit antiserum, 1:100)34 (link) and Cdk2 (Abcam, ab7954, 1:50). RingoA was detected using the R55A mouse monoclonal antibody12 (link), which recognizes the sequence NDHQSNK corresponding to amino acids 283-289 (hybridoma supernatant, 1:1). The slides were then washed in blocking buffer and incubated for 1 h at 37 °C with appropriate Alexa Fluor-labelled secondary antibodies (Invitrogen, A21442, A31571, A21441, A21468, A21203, A11017, 1:300). Finally, the slides were washed and mounted in ProLong Gold antifade reagent (Molecular Probes).
For telomere FISH staining, Telomere PNA FISH kit/cy3 (Dako, K5326) was used on 16 dpp testis spreads following the protocol described by the manufacturer.

For immunofluorescence staining, frozen tissue sections were cut at 7-8 μm, air dried and permeabilized with ice-cold methanol for 15 min. Sections were briefly washed twice with PBS and placed in 0.3% Triton X-100 10 min before blocking with 10% KPL (5140-0011, SeraCare) in PBS for 1 h at room temperature. The sections were incubated with primary antibodies against phospho-Stat3 (1:200; 9145, Cell Signaling Technology), insulin (1:200; PA1-26938, ThermoFisher), proinsulin (1:200; MAB-13361, R&D), Reelin (1:1000; AF3820, R&D), and GFAP (1:200; LS-B4775, LifeSpan Bioscience) at 4 °C overnight. Sections were washed three times with PBS and incubated for 1 h in the dark with the corresponding fluorophore-conjugated secondary antibodies, Alexa Fluor 488 Chicken anti-rabbit IgG (1:500; A-21441, Invitrogen), Alexa Fluor 488 Goat anti-guinea pig IgG (1:1000; A-11073, Invitrogen), Alexa Fluor 555 Goat anti-rabbit IgG (1:1000; A-32732, Invitrogen), Alexa Fluor 555 Goat anti-chicken IgG (1:500; A-21437, Invitrogen), Alexa Fluor 647 Goat anti-mouse IgG (1:500; A-21235, Invitrogen). Sections were counterstained using DAPI (1:1000 from 1 mg/ml; D9542; Sigma) to visualize cell nuclei and cover slipped with Fluoroshield (Sigma, F6182). Images were captured using the Olympus confocal microscope FV3000. ImageJ software was used to quantify MFI per islet area in the Il22ra1 x Ins2-cre animals.

For immunofluorescence experiments 2 × 10³ cells were seeded onto 8-well Lab-Tek chamber slides (Thermo Scientific, 177445) and grown for 48 h. Samples were fixed with 4% formaldehyde (252931 Panreac) for 15 min at RT, and permeabilized with 1 × PBS/0.1% Triton X-100 (Sigma-Aldrich) for 20 min at RT. Blocking was performed with 1 × PBS/10% BSA (A7906 Sigma-Aldrich) at RT for 1 h, followed by incubation with the corresponding primary antibody _ rabbit anti-H4K20me1 (ab9051, Abcam) or rabbit anti-H4K20me2 (ab9052, Abcam)_ at 1:1,000 dilution (see Supplementary Table 2) in antibody diluent (EnVision FLEX DM830 Dako), for 1 h at 4°C. Secondary antibody chicken anti-rabbit IgG Alexa Fluor 488 (A-21441 Invitrogen) at 1:500 dilution was incubated for 1 h at RT protected from light. Finally, slides were mounted using EverBrite mounting medium with DAPI (23002 Biotium). Immunofluorescence images were acquired with a Zeiss microscope, equipped with a 63X/1.4 NA immersion objective and an AxioCam MRm camera (Carl Zeiss). Fluorescence intensity measurements were performed using the ZEN lite software (ZEN lite 2.3 SP1, Carl Zeiss).

To verify the cellular content, primary hippocampal cultures were subjected to immunocytochemical staining on day 21 of culture development in vitro (DIV). The cultures were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by incubation with a solution of 0.2% Triton X-100/PBS for effective cell permeabilization. For immunofluorescence reactions, the cultures were then incubated for 2 h in the presence of a polyclonal mouse anti-GFAP (glial fibrillary acidic protein, a marker of differentiated astrocytes) primary antibody (Sigma-Aldrich G3893, Merck KGaA, Darmstadt, Germany, 1:500 dilution) and polyclonal rabbit anti MAP2 (a marker of differentiated neurons) primary antibody (Abcam 32454, Cambridge, UK, 1:500 dilution). Next, the cultures were subjected to a 45-min incubation in the following secondary antibody mixture: goat anti-mouse Alexa 647 (Thermo Fisher Scientific, A21236, Waltham, MA, USA, 1:800 dilution) and chicken anti-Rabbit Alexa Fluor 488 (Thermo Fisher Scientific, A21441, Waltham, MA, USA, 1:800 dilution). The stained material was observed using a Zeiss LSM 800 fluorescence confocal microscope (Carl Zeiss, Oberkochen, Germany).

HeLa cells plated on a 24‐well dish were fixed and permeabilized with 3.7% formaldehyde and 0.2% Triton X‐100. Cells were incubated in buffer containing 7.5 mg·mL⁻¹ glycine and then blocked with 3% bovine serum albumin and 0.1% Triton X‐100 for 30 min at room temperature. The cells were then incubated with anti‐FEZ1 (1 : 100, Atlas, HPA038490) and anti‐RAR (1 : 100, Abcam, ab28767) primary antibodies for 1 h at room temperature and then with chicken anti‐rabbit 488 (1 : 250, Thermo Fisher, A21441) and donkey anti‐goat 546 (1 : 250, Thermo Fisher, A11056) secondary antibodies for 40 min at room temperature. Nuclei were stained with 1 : 10 000 Hoechst 33342. Slides were examined with Leica confocal microscope.

After co-culturing of iNeurons and astrocytes for 38 days on chips and well plate, cells were washed with ice cold phosphate buffered saline (PBS, Gibco), fixated for 15 min at room temperature with 4% formaldehyde (Thermo scientific) and subsequently washed 3 times with PBS. Cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) solution for 10 min at room temperature. 5% goat serum (Sigma Aldrich) in PBS was used as a blocking agent for 1 h at room temperature. Primary antibodies (MAP2, Rabbit polyclonal, Abcam ab32454, 1:1000; Synapsin-1/2, Guinea Pig, Synaptic systems 106004, 1:500) were diluted in PBS with 1% goat serum and applied to the cells, which was then incubated overnight at 4 °C. Cells were washed 10 times for 1 min with PBS. Secondary antibodies (Chicken anti-Rabbit IgG Alexa Fluor 488, ThermoFisher A-21441, 1:500; Goat anti-Guinea Pig IgG Alexa Fluor 647, ThermoFisher A-21450, 1:1000) and DAPI (Thermo fisher) were diluted in 1% goat serum and added to the cells. Cells were incubated for 1 h at room temperature. Afterwards cells were washed 10 times for 1 min with PBS. Cells were imaged using a Zeiss LSM 510 confocal microscope at 10X magnification.