Antifade mounting medium

After following the above protocol, sections with dorsal hippocampal region were collected and separated in 12-well plate, washed with 0.1 M PBS, permeabilized with 0.25% Triton X-100, and blocked with 1.5% goat serum for 3 h at RT and then washed with 0.1 M PBS (pH 7.4) and incubated with mouse anti-COX IV (1 : 200) for 24 h at 4°C. The sections were then washed and incubated with Alexafluor-488 conjugated goat anti-mouse IgG (1 : 100) for 3 h at RT. Subsequently sections were treated with DAPI (Sigma) for 2-3 min. Finally, the sections were thoroughly rinsed thrice in distilled water, mounted onto gelatin-coated slides, and mounted in antifade mounting medium (Invitrogen). Fluorescence images were acquired on fluorescence microscope (Olympus, Model BX 51) and the mitochondrial distribution in CA1 and CA3 region was identified through intensity variation by Image J software in six random fields of 0.1 mm².

Control and SA treated mice were anesthetized and perfused initially with cold PBS followed by 4% paraformaldehyde. The brain was dissected out and kept overnight in the same solution. For cryosectioning brain was washed with PBS and then kept in 15% and 30% sucrose solution for 1 day each respectively. Coronal sections of brain were cut from the posterior side till midbrain using a cryotome (Thermo scientific) and kept in PBS in a 12 well plate. Sections were treated with 0.3% hydrogen peroxide in methanol for 30 min at −20 °C. Brain sections were kept in blocking buffer (2% FBS and 0.1% Triton X-100 in PBS) for 1 h. Following overnight incubation at 4 °C with primary antibody (1:500 in blocking buffer) cells were washed with the same buffer 3 times for 15 min each. Sections were incubated with appropriate secondary antibodies for 2 h at RT. Nuclei were stained with DAPI (1 μg/ml) for 2 min, washed and sections were mounted on glass slides using the antifade mounting medium (Invitrogen). Slides were observed under Nikon Eclipse 90i microscope (Nikon, Kawasaki, Japan).

The A2780 and A2780cp cells were grown on round glass coverslips (Fisher Scientific, Waltham, MA, USA) in 35 mm cell culture dishes. Following a 20 min fixation with pre-chilled methanol, the coverslips were washed with PBS, permeabilized with 0.2% Triton X-100-PBS for 15 min, and blocked with 2% bovine serum albumin-PBS for 30 min. The coverlips were then incubated with rabbit polycolonal p62 and goat polyclonal LC3 primary antibodies (1:50) at 37°C for 90 min in the dark, followed by three 10 min washes in PBS. Subsequently, the coverlips were incubated with goat-anti rabbit IgG-TR and donkey anti-goat IgG-FITC secondary antibodies, respectively (1:1,000) at 37°C for 1 h in the dark, and washed three times (10 min/wash) with PBS. All of the antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The coverslips were mounted onto glass slides, with antifade mounting medium purchased from Invitrogen (Paisley, UK). The images were captured using a Zeiss Observer.Z1 microscope (Zeiss, Oberchoken, Germany) and Slidebook 4.2.0.11 computer software (Intelligent Imaging Innovations, Inc., Denver, CO, USA).

13 mm round glass slips (VWR) were prepared in 24 well plates by sterilising in 70% ethanol before monocyte seeding. PBMC separated monocytes were seeded in these 24 well plates and cultured for 7 days as described above. On day 7 DC on the glass cover slips (VWR) were washed, stained with DAPI (Thermo Scientific) and mounted with antifade mounting medium (Invitrogen) on a glass slide (Corning). Slides were left to dry for at least 24 hours in a dark area before transfer to a fridge. Imaging was performed on Zeiss LSM800 AiryScan confocal microscope using APC (ex651-em660) and DAPI (ex353-em465) channels with Plan-Apochromat 40x/1.30 Oil M27 objective. Image analysis was performed on Zen 2 software.

IF detection of marker proteins in cells, myotubes, neurospheres or neurosphere-like structures derived from cancer cells was performed exactly as described [11 (link)]. Primary antibodies were Myc (Cell Signaling, #13987. 1:400), Msi1 (Cell Signaling, #5663. 1:200), Sox1 (Abcam, #ab109290. 1:250), Myod1 (Novus Biologicals, #NB100-56511; Cell Signaling, #13812. 1:200), Oct4 (Cell Signaling, #83932. 1:500), Pax3 (Abcam, #ab15717. 1:500), Myoglobin (Cell Signaling, #25919. 1:200), Myosin heavy chain (R&D systems, #MAB4470. 1:200), Map2 (Cell Signaling, #8707. 1:200), Synapsin-1 (Cell Signaling, #5297. 1:200). Secondary antibodies were anti-mouse IgG (FITC-conjugated) (Sigma-Aldrich, #F9137. 1:1,000), Alexa Fluor R 568 donkey anti-rabbit IgG (H+L) (Invitrogen, #A10042. 1:1,000), and Cy3-AffiniPure donkey anti-goat IgG (H+L) (Jackson ImmunoResearch Labs, #705-165-147. 1:1,000), anti-mouse or rabbit Alexa Flour 594 (ThermoFisher Scientific, #A21207, #A21203. 1:500), anti-mouse or rabbit Alexa Fluor 647 (ThermoFisher Scientific, #A31573, #A31571. 1:500). Cell nuclei were counterstained with DAPI. Afterwards slides were rinsed, and coverslips were mounted with anti-fade mounting medium (Invitrogen, #S36936). Cells were viewed with a fluorescence microscope (Zeiss LSM 880).