Fluoro gel

Freshly isolated femurs were fixed in 4% paraformaldehyde overnight, followed by 1 to 3 days decalcification in 10% EDTA. For paraffin section, bone samples were processed with Sakura Tissue Tek VIP 5 Tissue Processor (Sakura America, Torrance, CA), and paraffin sections were cut in 5um thickness. Sections were deparaffinized with xylene, followed by Alcian Blue-Hematoxylin-Orange G staining. For frozen section, bone samples were processed with the CryoJane tape-transfer system. Sections were blocked with Power Block™ Universal Blocking Reagent for 30 min to 1 hr and then stained overnight with anti-BrdU (GE Healthcare RPN202 1: 100), rabbit-anti-Aggrecan (Millipore, 1:300), rabbit-anti-Perilipin (Cell Signaling, 1:300) and goat-anti-Osteopontin (R&D, 1:300). Donkey-anti-goat Alexa Fluor 488 and Donkey-anti-goat Alexa Fluor 647 were used as secondary antibodies (all from Invitrogen, 1:300). Antibodies were diluted with Antibody Diluent Solution (Invitrogen 00–3218). Slides were mounted with FLUORO-GEL (Electron Microscopy Science 1798510), and images were acquired with an Olympus slide scanner.

Paraffin sections were incubated overnight at 60°C before being deparaffinized with three changes of Xylene for 10 minutes each, and rehydrated with isopropyl alcohol at two changes of 100% alcohol, two changes of 95% alcohol, one change of 75% alcohol, and one change of distilled water for 1 min each change. Slides underwent heat induced epitope-retrieval in Tris Urea solution. After rinsing with Tris Buffered Saline solution three times, an ImmEdge ™ Hydrophobic Barrier pen (Vector Laboratories, Inc., Burlingame, CA) was used to isolate tissue sections and slides were submerged with blocking buffer for 1 hour in a humidified chamber (Bobeck et al. 2015 (link)).
To stain IL-10, tissues were coated in rabbit aIL-10 polyclonal antibody (Bioss Inc., Boston, MA) at 1:300 dilution in blocking buffer overnight. To stain IFNγ, a contiguous intestinal section was coated in rabbit anti- chicken IFNγ polyclonal antibody (My Biosource Inc., San Diego, CA) at 1:100 dilution in blocking buffer overnight at 4°C in a humidified dark enclosure. Slides were stained with 1:100 diluted Donkey anti-rabbit Dylight®594 (Bethyl, Montgomery, TX) for one hour in a humidified chamber. Nuclei were highlighted by 4',6-diamidino-2-phenylindole (DAPI) in Fluoro-Gel with tris buffer solution (Electron Microscopy Sciences, Hatfield, PA).

Immunofluorescence was performed based on our previous protocol (Shetty et al., 2016 ). Briefly, cervical cancer cells (HeLa and SiHa) were cultured on 8-chamber plates, treated with Nef (25μM), and incubated at 37°C and 5% CO₂ for 48 hrs. Following treatment cells were fixed (4% paraformaldehyde) for 15 minutes and blocked with 5% goat normal serum (Invitrogen) with 0.3% Triton X- 100 (Sigma–Aldrich) in PBS. Cells were washed (PBS) and incubated with primary antibodies to pH2AX (1:200), caspase-3 (1:200) and cytochrome c (1:200) respectively for overnight at 4°C. After three successive washings, cells were probed with 0.1μg/ml of secondary anti-mouse Ig-G or anti-rabbit Ig-G conjugated with FITC for 1 hr at RT. Cells were counter-stained with DAPI (30nM) for 10–15 minutes with PBS, and a coverslip with Fluorogel (Electron Microscopy Sciences, PA, USA) was prepared for visual inspection with an Olympus Fluoview laser scanning confocal microscope.