Sodium citrate ph 6

Germ cells were spread out on a slide, dried for 2 h at room temperature, rehydrated in PBS 1× for 5 min and incubated for 40 min at 95°C in 10 mM sodium citrate pH 6 (Sigma), 0.05% Tween 20 (Carlo Erba). Next, they were permeabilized with 0.1% Triton-X at room temperature for 30 min. After blocking in 5% dried milk and washing in PBS 1×, cells were incubated in 1% dried milk for 1 h at 37°C in a humidified chamber using the following antibodies: anti-HA 1/800 (Roche 1867423), anti-H3 1/200 (Millipore 05-928), anti-H4 1/200 (Abcam ab10158), anti-γH2AX 1/500 (Abcam ab81299), anti-H3K9me3 1/600 (Abcam ab8898), anti-H3.3 1/100 (Millipore 09-838). After washes in PBS 1× for 30 min at room temperature, a secondary cyanine 2 or cyanine 3 antibodies were applied for 30 min at room temperature in 1% fetal bovine serum, PBS 1×. All samples were incubated for another 5 min in Hoechst 33342 (Invitrogen H3570) to counterstain DNA.
Of note, immunostaining with the 1/100 CD9 (BD pharmagen 553758) antibody was performed immediately drying the germ cells for 2 h at room temperature and their rehydratation in PBS 1× for 5 min. The slides were then blocked with 10% bovine fetal serum for 1h at room temperature and subsequently incubated in humidified chamber with the antibody for 1 h at 37°C.

Tissue specimens were fixed in 10% formalin for 12 to 24 h, dehydrated, and paraffin embedded. Liver sections were stained with H&E following standard protocols. For immunohistochemistry, sections were subjected to antigen retrieval by boiling the slides in sodium citrate pH 6 (Sigma Aldrich) for 15 min and then permeabilized in phosphate-buffered saline (PBS) with 0.25% TritonX-100 for 5 min. Subsequently, after 10 min incubation at room temperature in protein blocking solution (Dako, Glostrup, Denmark), sections were incubated at 4 °C for 48 hours with the anti-8-Hydroxyguanosine antibody (LifeSpan Bioscience Inc, Seattle, Washington, USA). Sections were washed in PBS for 15 min and incubated for 25 min at room temperature with DAKO real EnVision detection system Peroxidase/DAB + (Dako) according to manufacturer’s instruction. Coverslips were mounted with Permount and evaluated under a light microscope. To evaluate lipid accumulation, serial 4.5 μm cryosections from liver specimens embedded in OCT compound (Tissue-Tek Sakura, Torrance, CA) were stained with Oil Red O (Sigma-Aldrich) and hematoxylin to counterstain nuclei.

Tissue specimens were fixed in 10% formalin for 12–24 h, dehydrated, and paraffin embedded. 4 µm thick sections were stained with hematoxylin-eosin (H&E) following standard protocol. For antigen retrieval slides were boiled in sodium citrate pH 6 (Sigma Aldrich, Milan, Italy) for 15 min. Sections were permeabilized in phosphate-buffered saline (PBS) with 0.25% TritonX-100 for 5 min and then were incubated at room temperature in protein blocking solution for 10 min (Dako, Glostrup, Denmark) and overnight at 4 °C with the primary antibodies (anti-pcna 1:100, Santa Cruz Biotechnology, Santa Cruz, CA; or anti-PTEN 1:2000, Abcam, Cambridge, UK or anti-SMAD4 1:400, Abcam, Cambridge, UK). Sections were rinsed in PBS (15 min) and incubated at room temperature with DAKO real EnVision detection system Peroxidase/DAB⁺ (25 min, Dako, Glostrup, Denmark) according to manufacturer’s instruction. Image analysis was made through Image J software. For each sample, 10 representative images were acquired with a 20x objective. The percentage of stained area/total area was evaluated. Values from all consecutive images for each sample were averaged. For negative controls, 1% nonimmune serum in PBS substituted the primary antibodies.