Slowfade gold

Adventitial and intimal T cells in aortic root, arch, and thoracic descending aorta were analyzed by en face confocal microscopy, as previously described [27] (link). Mice were perfused with PBS and heparin, and aortic segments harvested, opened longitudinally to expose the lumen, and post-fixed for 30 min at 4°C in 2% paraformaldehyde. Periadventitial fat was removed, and tissues were blocked for 1 h with anti-CD16/CD32 Fc-block (BD Biosciences) and 10 µg/ml non-immune mouse IgG (Sigma). DAPI, anti-CD3e-FITC, and either anti-γδTCR APC or anti-CD4 APC (RM4-5) were added directly to the blocking solution, and the tissues incubated overnight at 4°C. Tissues were then washed with PBS, mounted between two glass coverslips with SlowFade Gold (Molecular Probes), and imaged using a Leica SP2 confocal microscope. T cells were located by scanning the aortic tissue by eye for green fluorescence in the FITC filter, which was easily distinguishable from yellow non-specific autofluorescence. To assess the specificity of far-red fluorescent images obtained with the confocal microscope, we verified that a corresponding orange-red signal was not seen in the fluorescence microscope using the Cy3 HQ filter cube.

Cells were washed twice with PBS and fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature, spread on slides, and permeabilized with 0.05% Triton X-100. After incubation in PBS containing 4% BSA overnight, cells were incubated for 45 min with anti-aldolase rabbit serum (Aldo; 1:1,000; gift from P. Michels, Edinburgh, UK). After washing with PBS, samples were incubated for 45 min with a secondary anti-rabbit IgG antibody conjugated to Alexa Fluor 594 (Thermo Fisher Scientific, Waltham, MA, US). Slides were washed and mounted with SlowFade Gold (Molecular Probes). Images were acquired with MetaMorph software (Molecular Devices, Sunnyvale, CA, US) on a Zeiss Imager Z1 or an Axioplan 2 microscope as previously described [42 (link)].

WT, CYP51^RNAi non-induced and induced cells were fixed in 3% paraformaldehyde in PBS for 2 minutes. After PFA fixation (3%), glycine was added (100 mM in PBS, 10 min) and cells were spread on poly-L-lysine-coated slides. Cells were permeabilized with Triton-X100 0.2% in PBS for 10 min and washed once in PBS. Samples were incubated with the primary antibody for 1 hour at room temperature in a moist chamber: anti-PFR (mouse monoclonal L8C4, neat, a kind gift from Pr. K. Gull, Oxford University). After two PBS washes, cells were incubated for 45 min with the secondary antibody anti-mouse conjugated to FITC (Sigma F-2012, 1:100 dilution). Nuclei and kinetoplasts were labeled with DAPI (10 μg.mL^-1 in PBS for 5 minutes) and washed twice in PBS for 5 minutes. Slides were mounted with Slowfade Gold (Molecular Probes S-36936).

The formalin fixed samples were embedded in paraffin and processed using standard histological procedures, stained with haematoxylin and eosin, followed by routine histological examination. All organs samples were examined for the presence of ISAV by immunohistochemistry (IHC) as previously described [18 (link)] using rabbit antibodies to recombinant ISAV nucleoprotein (NP) [19 (link)] and scored using our previously described IHC ISAV scoring system [6 (link)]. Immunofluorescent labelling (IFAT) was performed on acetone fixed cryo-sections as previously described [8 (link), 20 (link)] using a monoclonal antibody to ISAV haemagglutinin esterase (HE) [20 (link)] and Alexa Fluor^® 488 conjugated anti-mouse IgG (Life technologies) for detection. Sections were mounted in SlowFade^® Gold (Molecular Probes), and were examined with a Zeiss LSM 710 confocal laser scanning microscope (Carl Zeiss, Germany) using a 63× oil immersion objective and scored [6 (link)].

Third instar wandering larvae were dissected in Schneider’s medium and fixed in 4% PFA for 25 mins followed by washing with 0.1% PBST for 20 mins at RT. Brains are then washed twice with 1XPBS for 2 min. The terminal transferase (TdT) reaction was performed as follows: First brains were treated with TdT reaction buffer and incubated for 10 min followed by treatment of freshly made TdT reaction buffer cocktail (TdT reaction buffer, 5-Ethynyl-dUTP, TdT) for 60 min at 37°C and at 500rpm. After the TdT reaction, brains were washed twice with 3% BSA at RT for 5 min. Then Click-iT reaction was performed by adding click iT reaction cocktail (Click-iT reaction buffer, Click-iT reaction buffer additive) for 30 min at RT. At this step samples were protected from light. Brains were washed with 3% BSA twice for 5 min after removing Click-iT reaction buffer cocktail and then incubated with Hoescht (1:1000) for DNA staining followed by washing with 1XPBS for 10 min. Samples mounted in Slow-Fade Gold (Molecular probes) and subsequently imaged using Zeiss LSM710 with a 40x/1.4NA oil objective and DPSS laser (561nm). Induction of apoptosis by the UAS-Hid line driven by pnt-Gal4 was used as positive control. TUNEL positive nuclei were counted and plotted using GraphPad Prism 5 software.

For immunofluorescent staining, male Sprague-Dawley rats were subjected to FUS-BBB treatment as described and sacrificed 2 hours after the last sonication session. EB dye was IV-injected as a bolus immediately after ultrasound exposure to serve as an indicator of BBB opening. The animals were anesthetized with isoflurane, and then euthanized with isoflurane overdose. They were then perfused transcardially with 0.9% normal saline, followed by 4% paraformaldehyde. The brains were harvested and put in sucrose for frozen sectioning. Frozen sections (thickness = 20 μm) were incubated for 1 hour in 0.3% BSA in PBST at room temperature. Anti-GFAP antibody (1:100; #AB5804, Millipore) was diluted in a blocking buffer and incubated at 4 °C overnight. The appropriate fluorophore-conjugated secondary antibody was added in a 1:200 dilution in the blocking buffer and incubated for 90 min at room temperature before mounting with DAPI (5 g/ml) and SlowFade Gold (Molecular Probes). Apoptosis in sections were assessed by the TUNEL assay using an In situ cell death detection kit according to manufacturer’s instructions (Roche). Immunofluorescent images were viewed with a fluorescence laser-scanning confocal microscope (Olympus FV10i, Center Valley, PA). The immunofluorescent image acquisition for the DAPI, Rhodamine and FITC channels were kept constant over all samples.