Dapi nuclear stain

The cells were grown on 24 well-plates as described above. After 4 h and 24 h PI with H. parasuis, the cells were fixed with 4% paraformaldehyde, macrophages were labelled with DAPI (nuclear stain) (Sigma-Aldrich) and with Alexa Fluor (AF) 594 conjugated phalloidin (Life Technologies) to visualize the actin cytoskeleton. Microscopy was performed using an epifluorescence inverted microscope Olympus IX51 equipped with a LUCPlanFLN 40 × (NA 0.60) objective using fluorescence mode to detect DAPI and AF-594.

Mice were anesthetized with pentobarbitone (50 mg, intraperitoneally) and transcardially perfused with 4% paraformaldehyde (0.1 mol/L PBS). A vibrating microtome (Leica VT1200) was used to obtain 80-μm sections of brain, spinal cord, and liver. Sections were dehydrated by incubation in 50% ethanol in distilled water (v/v) for 30 min and then were washed three times in 0.3 mol/L PBS, followed by incubation in 10 mM sodium citrate (pH 6, 85°C, 30 min) for antigen retrieval. Sections were then incubated in the blocking solution (5% normal goat serum in 0.3mol/L PBS with 0.3% Triton X-100) for 1 hr at room temperature. Samples then were incubated for 48 hr on a shaker at 4°C with the following primary antibodies: rabbit anti-Myc (Abcam, ab9106), mouse monoclonal anti-MeCP2 (Sigma, WH0004204M1), and chicken anti-GFP (Abcam, ab13970). The primary antibodies were then washed off (3× 0.3 mol/L PBST), and secondary antibodies were applied to the sections overnight at 4°C: Alexa Fluor 488 goat anti-mouse/rabbit (Invitrogen; 1/500), Alexa Fluor 546 goat anti-mouse/rabbit (Invitrogen; 1/500), Alexa Fluor 649, goat anti-mouse (Jackson ImmunoResearch Laboratories, 112-495-003JIR). Finally, sections were incubated with DAPI nuclear stain (Sigma; 1/1,000) for 30 min at room temperature before mounting with Vectashield (Vector Laboratories).

Sections from two regions of the heart (heart base and apex) were examined. Sections of each subgroup (dose rate, age) were stained together with samples from sham-irradiated control animals and negative staining controls.
Specimens were stained with hematoxylin/eosin (HE) and sirius red following standard protocols. For specific immune fluorescence staining, sections were fixed in ice-cold ethanol/acetone, 50/50, for 10 min. Specimens were then washed with PBS (3 min) and incubated in blocking solution (PBS, bovine serum albumin 2%) for 20 min. Primary antibodies or isotype specific Ig (negative staining controls) were applied for 1 h diluted in blocking solution. Specimens were washed again, and corresponding, fluorescence-labeled, secondary antibodies were added (Table 2). After 45 min, specimens were rinsed again in PBS and incubated with DAPI nuclear stain for 5 min (Sigma Aldrich, Saint Louis, USA). Sections were embedded in Mowiol 4-88/DABCO (Carl Roth GmbH, Karlsruhe, Germany). Lectin-stained heart sections (fluroescein lycopersicon esculentum tomato lectin, FL-1171, Vector laboratories) were kindly provided by Fiona Stewart’s laboratory, NKI (The Netherlands Cancer Institute).

Vero 76 and BHK cells were seeded into 24-well plates and infected at MOI 0.01. Infected cells were fixed and permeabilised (4% paraformaldehyde 20 min, 0.1% Triton X-100 for 10 min) at 24, 36, 48, 60 hours post infection and stained for 1 h at room temperature with mouse anti-E monoclonal antibodies 3.91D [56] (link), followed by 30 min staining with the secondary Alexa Fluor 488 goat anti-mouse antibodies and DAPI (nuclear stain, Sigma-Aldrich), with intercalating 3×5 min washes with PBS between each step. After a final wash with PBS, cells were imaged using IN-cell 1000 analyser (GE Healthcare) at the required excitation and emission wavelengths and the percentage of E-positive cells (% infected) was determined using IN-Cell 1000 Workstation software where: % infected cells = [(number of cells at 480 nm)/(number of cells at 360 nm)]_KUNV×100−[(number of cells at 480 nm)/(number of cells at 360 nm)]_mock×100.

HepG2 cells constitutively expressing LDLR-GFP were obtained from Peter Tontonoz (University of California, Los Angeles). Cells were resuspended in 10% lipoprotein-deficient serum (LPDS), plated in chamber slides (LabTekII; Thermo Fisher Scientific) and transfected with miRNA mimics (80 nM, Dharmacon) using Lipofectamine® RNAi MAX (Invitrogen). After 48 h, the cells were washed and fixed with 4% paraformaldehyde and stained with DAPI nuclear stain (D-9542, Sigma-Aldrich) for 10 min. After washing, cells were mounted with coverslips using mounting medium for fluorescence (H-1000; Vector Laboratories Inc., Burlingame, CA, USA). Fluorescent images were collected with an LSM 510 confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany) with 63 × /1.4 oil objective. The frame size was 1,024 × 1,024. The manufacturer's software was used for data acquisition and ImageJ for fluorescence profiles. The weighted colocalization coefficients were calculated using AIM (Carl Zeiss). The cells were visualized with an Axiovert 25 (Carl Zeiss) with a 10 × /0.25 or 32 × /0.40 objective.

Antigen retrieval was performed in citrate buffer (pH 6.0) in a pressure cooker. Tissues were blocked in CAS block (Thermo Fisher Scientific) and incubated overnight with primary antibodies diluted in CAS block (Table 1):
Slides were washed 3 times in PBST, incubated overnight with secondary antibodies, and diluted in PBS/BSA 1% (Table 2):
Slides were then washed once in PBS and closed with cover glass using ImmuGio mounting medium (DiaSorin, 2505-5). Relevant tissue samples were incubated with DAPI nuclear stain (Sigma-Aldrich, D9564; diluted 1:100 in double distilled water [DDW]) for 5 minutes, then washed 5 minutes in DDW before closing. Pictures were taken with Nikon confocal microscope, and the software NIS-Elements. For UCN3 fluorescence analysis, the same microscopy and NIS software settings were used per experiment, and fluorescence measurements were taken using ImageJ (NIH) (RGB measurement Plugin).