Triton x 100

Human umbilical vein endothelial cells (HUVECs) were seeded onto 12 mm glass coverslips in 24 well plates at 75,000 cells/well. After adhering, cells underwent pretreatment with 17R-RvD1 (10 nM, 100 nM) or benzo-RvD1 (10 nM, 100 nM) or vehicle for 30 minutes, followed by addition of TNFα (1 ng/mL, Sigma-Aldrich) for 2 hours. Coverslips were then washed in ice-cold phosphate buffered saline (PBS), then fixed in 4% formaldehyde and treated with 0.5% Triton X-100 (Sigma-Alrich). Blocking with 2% FBS in 0.3% Triton X-100 was performed before overnight incubation at 4°C with anti-p65 antibody (1:50; Santa Cruz Biotechnology, Dallas, TX). An Alexa Fluor 488 tagged secondary antibody (1:200, Life Technologies, Carlsbad, CA) was then used, followed by DAPI nuclear counterstaining. Images were taken with a fluorescence microscope and analyzed with ImageJ. NF-κB activation was quantified by the ratio of fluorescent intensity in the nuclear vs. cytoplasm.

The cells, treated for 72 h with the OCF as reported above, were detached with Accutase (Euroclone, Milan, Italy), then washed twice in PBS and resuspended in 4% PFA in PBS. Following an incubation at 4 °C for 10 min, the cells were centrifuged at 400× g for 5 min and the pellet was resuspended in PBS 0.25% Triton™ X-100 (Sigma Aldrich, Milan, Italy) for 5 min. The cells were pelleted again and incubated O/N at 4 °C with a 2 µg/µL anti-p53 mouse primary antibody (1:100, Santa Cruz Biotechnology, distributed by DBA, Milan, Italy) in PBS 0.1% Triton™ X-100 0.2% BSA. The cells were then washed in PBS twice and incubated for 1 h at RT with an anti-mouse antibody conjugated with FITC (1:200, Merck Millipore, Milan, Italy) in PBS 0.1% Triton™ X-100 0.2% BSA. The cells were washed twice in PBS, resuspended in PBS and analyzed with a BD FACSCanto II flow cytometer.

EdU detection was performed using the Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen). Sections were permeabilized with 0.2% Triton X-100 (Progen Industries) in dPBS for 30 min, washed with 3% protease-free BSA (Sigma-Aldrich) in dPBS (2 × 5 min), then incubated with 300 μl reaction mixture prepared according to kit directions for 30 min. In the first pilot run, cells were not double labeled with Brn3a to distinguish RGCs, because at P0 there are as yet no displaced amacrine cells in the GCL (Perry, 1981 (link)). In the more extensive follow-up experiment we also wished to obtain laser-captured E18 RGCs from P5 retinas at a time when their axons are in the process of innervating the SC; however, at this age potentially EdU⁺ amacrine cells are now present in the GCL. Thus for all groups in this second series we additionally immunostained retinal sections for Brn3a protein, an established marker for RGCs projecting to the SC (Nadal-Nicolás et al., 2009, 2012 (link)). Retinal sections were washed with dPBS (2 × 10 min) and incubated overnight at 4°C with anti-Brn3a goat primary antibody (AB; Santa Cruz Biotechnology, SC-31984) 1:100 in antibody diluent (10% normal horse serum and 0.2% Triton X-100 in dPBS). After washes, sections were incubated in donkey anti-goat Cy3 (Jackson ImmunoResearch, 705-166-147; 1:200 in antibody diluent) for 2 h.

Livers were removed from euthanized animals at the end of experiment and post-fixed in 4% PFA at 4°C overnight. Then, 5-μm sections were obtained, submitted to permeabilization for 2 h with 0.4% Triton X-100 (Santa Cruz, Cat. #sc-29112) and blocking (1% BSA for 2h in ambient conditions). Each section was next sequentially stained with primary (72 h at 4°C) and secondary (2 h in ambient conditions) antibodies. After PBS washes, the slides were dehydrated, followed by mounting with the VECTASHIELD mounting medium. Imaging was performed under a Leica confocal microscope.

Clinical liver cancer tissues and paired non-cancerous tissues were fixed in formalin at room temperature for 24 h, embedded in paraffin and cut into 5-µm consecutive sections. Following deparaffinization and antigen recovery in a sodium citrate solution (pH 6.0) for 20 min at 98°C, the sections were washed thrice with 0.01 mol/l PBS for 5 min each time, blocked for 1 h in 0.01 mol/l PBS containing 0.3% Triton X-100 (Santa Cruz Biotechnology, Inc.) and 5% BSA (Gibco; Thermo Fisher Scientific, Inc.), and incubated with an anti-BUB1 antibody (cat. no. ab195268; 1:200; Abcam) overnight at 4°C. Following washing with 0.01 mol/l PBS, the sections were incubated with 0.01 mol/l PBS containing a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody (cat. no. ab6759; 1:500; Abcam) for 2 h at room temperature, followed by development with 0.003% H₂O₂ and 0.03% 3,3′-diaminobenzidine in 0.05 mol/l Tris-HCl (pH 7.6). The categories and percentages of immunohistochemical stained cells were assessed in five independent high-power microscopic fields for each tissue sample using a light microscope (magnification, ×400).