Bx61w1 microscope

To visualize phagocytosis under conditions of proinflammatory LPS and PAL stimulation and its modulation by 5-AZA and SAM methylation modulators, we used confocal microscopy. SIM-A9 cells were seeded on sterile coverslips in six-well plates covered with 0.01% poly-L-lysine solution (Sigma, p4707). After treatment, cells were fixed by adding 4% paraformaldehyde (PAF) (Sigma, 158127) for 10 min, washed three times with 1 × PBS, and were permeabilized with PBS solution 1 × + 0.1% Triton X-100 (PBST) (Sigma, × 100) for 10 min. Next, the cells were blocked in PBST + 10% goat serum (GIBCO, 16210064) for 30 min and washed three times with 1 × PBS. Microglia immunodetection was performed by overnight primary antibody anti-mouse Iba-1 (1:200) (Abcam, ab178847) incubation at 4°C followed by goat anti-rabbit IgG coupled to Alexa Fluor 546 (1:1000) (Invitrogen, A-11035). Finally, cells were washed 3 × with 1 × PBS and mounted in VECTASHIELD mounting medium with DAPI (Vector Laboratories, H-1000-10). Fluorescent signals were detected by confocal-laser microscopy using an Olympus BX61W1 microscope with an FV1000 module with diode laser. Finally, the images were processed with ImageJ software.

A K-sensitive electrode was coupled with a double-barreled LFP recording electrode, creating a K-LFP recording electrode. The double-barreled electrode was filled with saline and cemented to the K-sensitive electrode such that the distance between the tips of the electrodes was approximately 50 μm apart [22 (link)]. First, the K-sensitive electrode was mounted to a head stage of an Axopatch 200B amplifier. A differential reference electrode for the head stage was inserted into a chamber of the double-barreled LFP electrode; the other chamber was used to record the extracellular LFP connected to a head-stage of an Axopatch 200B amplifier. This latter signal was differentially recorded from a common ground wire, attached to the scalp. All amplifiers were then digitized (Digidata 1440, Molecular Devices). LFP and extracellular K signals were low-pass filtered at 5 kHz. Electrodes were lowered into the right somatosensory cortex in steps of 0.1 mm under an Olympus BX-61W1 microscope with 4X PlanN objectives.

For the analysis of trimeric interaction, the BiFC-FRET assays were performed in HEK293 cells maintained under standard cell culture conditions. Cells were seeded on 6-well plates, 48 h later transfections were carried out using 1 µL of PEI (40 kDa) 15 mM (Sigma-Aldrich, Saint Louis, MI, USA) for each microgram of DNA. Trimeric interactions by BiFC-FRET were performed co-transfecting the VC-, VN- and EFCP- constructs. BiFC-FRET image acquisition was done 48 h after transfection in an Olympus BX61W1 microscope; 10 nm size photographs were collected in spectral mode (420–660 nm) using 10 nm of stepsize under the confocal parameters 600v, 1X gain, 0% offset, and 10% laser potency with 20X objective. The BiFC-FRET quantification (E-value) was performed using ImageJ and the FRETTY plug-in. This plug-in uses 2D deconvolution spectral unmixing by comparing the donor and acceptor images to measure the energy transfer between Venus and ECFP [69 (link)]. For all assays, three independent experiments were performed. pBiFC-bJun-VN173, pBiFC-bJun-YN173, pBiFC-bFos-VC155, pBiFC-bFos-YC155, pBiFC-bFosΔZIP-VC155, pFlag-p65-Cerulean and pFalg-p65Δ25-Cerulean expression vectors used for BiFC-FRET standardization were kindly provided by Hu Chang-Deng [26 (link)].

For widefield imaging, a white light source (LED Engin LZ1-10CW00) was used for illumination. Illumination light and fluorescence signals were filtered through a GFP filter cube (460/50 excitation, 540/50 emission). A 4× 0.13 NA objective (Olympus) and a Photometrics Evolve 512 Delta camera were used to collect the emitted light. The field of view was roughly 5.5 × 5.5 mm². The frame rate was 20 Hz.
Two-photon Ca²⁺ imaging was performed with an Ultima system (Prairie Technologies) built on an Olympus BX61W1 microscope. A mode-locked laser (Coherent Chameleon XR Ti:Sapphire) tuned to 950 nm was raster scanned at 5 Hz for excitation while emitted GCaMP6s fluorescence was collected through a green filter (525/70 nm). Laser power at the sample was 20–80 mW. Dwell time was set to 2 μs. To increase imaging speed, resolution along the y-axis was reduced by a factor of 4. The final pixel size was 0.9 × 3.6 μm. A 20× 1.0 NA objective (Olympus) was used to yield a 465 × 465 μm² field of view. Imaging depths were between 150–350 μm.

C57/BL6 mice were dosed with isoquercetin twice per day by oral gavage, once in the morning and once in the afternoon. This was performed for 48 hours prior to commencement of the experiment, with one final dose in the morning of the experiment. Mice were anaesthetized by intraperitoneal ketamine (125 mg/kg), xylazine (12.5 mg/kg), and atropine (0.25 mg/kg) injection, and maintained with pentobarbital (5 mg/kg) as required, through a carotid artery cannula. The cremaster muscle of the testicle was affixed over a glass slide, and platelets were labeled with DyLight 649 anti-GPIbα antibody (0.2 μg/g mouse weight). Arteriole walls were injured using a Micropoint ablation laser unit (Andor Technology PLC, Belfast, Northern Ireland), with thrombus formation observed using an Olympus BX61W1 microscope (Olympus Corporation, Tokyo, Japan). Images were captured prior to and after injury using a Hamamatsu digital camera (C9300 charge-coupled device camera; Hamamatsu Photonics UK Ltd, Hertfordshire, United Kingdom) in 640 × 480 format. Images were analyzed using Slidebook 6 software (Intelligent Imaging Innovations, Colorado, United States). Experiments in mice were performed under a UK Home Office license after approval from the University of Reading Local Ethical Review Panel.