Alexa fluor 647 donkey anti mouse

HeLa cells were transfected with equal molar amounts of MHC-I-V_N-Flag and Nef-V_C. Twenty-four hours post-transfection cells were washed with ice cold PBS, and subsequently anti-HLA-A2 (BB7.2; Biolegend) antibody was added at a dilution of 1:300. Antibody was allowed to bind for 20 minutes at 4 °C. Following antibody binding, cells were washed 3X with cold PBS, and either fixed (time 0 minutes), or supplemented with warm complete media and incubated for 90 minutes at 37 °C. Cells were then fixed, permeablized, and immunostained with donkey anti-mouse AlexaFluor 647 (1:400; Jackson ImmunoResearch) secondary antibody for 2 hours to detect the internalized antibody. Coverslips were then washed 3X in PBS and mounted using DAPI-Fluoromount-G (Southern Biotech).

Aortic tissue was harvested from all animals. AAA tissue was fixed in Histochoice (VWR), and paraffin embedded. Paraffin blocks were sectioned at 5 μm, and deparaffinized. Processing for antigen retrieval was performed with Sodium Citrate solution, pH 6.0, for 10 min. Tissue sections were blocked with 10% serum, and sections were incubated with primary antibody anti-CD68, 1:100 (Bio-Rad, MCA341GA), CCR2, 1:200 (Novus-Bio, NBP1-48338), CD86 (Thermo-Fisher, #942-MSM-P0) and CD206 (Thermo-Fisher, #187004-1-AP). For Immunofluorescence, sections were incubated with donkey anti mouse (Alexa Fluor 647), and donkey anti rabbit (Cy3) (Jackson ImmunoResearch Laboratories). All sections were imaged using a Leica THUNDER Imager 3D tissue microscope system. Sections were then incubated with anti-mouse secondary antibodies conjugated with HRP (Cell Signaling), DAB peroxidase substrate kit (Vector Laboratories), and counter stained with hematoxylin, imaged using an Olympus fluorescent microscope system. To evaluate AAA tissue morphology and pathology, tissue sections were also evaluated using Hematoxylin and Eosin (H&E), Mason Trichrome (MT) and Verhoeff-Van Gieson (VVG), imaged using NanoZoomer, Supported by the Alafi Neuroimaging Laboratory. All AAA wall staining intensity was analyzed in 3 random ROIs, quantified with Image J, and normalized to the total area of the aortic wall media.

The samples were fixed using 4% formalin for 20 min, washed 3× with PBS, and blocked overnight in CB buffer (PBS supplemented with 50 mM NH₄Cl, 0.1% Saponin, and 2% BSA (IgG-free, Protease-free)). The cells were stained for the NP protein of IDV using rabbit anti-IDV NP (Genscript, 1:1000 in CB buffer) or mNeonGreen using mouse anti-mNG (Chromotek, 1:500 in CB buffer) for 2 h at room temperature, followed by secondary antibodies donkey anti-Rabbit Alexa Fluor 594 (Jackson ImmunoResearch, 1:400 in CB buffer), donkey anti-Mouse Alexa Fluor 647 (Jackson ImmunoResearch, 1:400 in CB buffer), or donkey anti-Rabbit Alexa Fluor 647 (Jackson ImmunoResearch, 1:2000 in CB buffer) for 1h. The samples were counterstained using 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, Waltham, MA, USA) and visualized using a Cytation 5 Cell Imaging Multimode Reader (Agilent BioTek, Sursee, Switzerland) equipped with 4× (numerical aperture (NA): 0.13), 10× (NA: 0.3), and 40× (NA: 0.6) air objectives. Images were further processed with Fiji (v1.53q); the brightness and contrast of all images were adjusted identically to the corresponding controls [27 (link)]. Figures were generated using the FigureJ plugin (v1.36) [28 (link)].

Cultures (DIV 18–20) were washed with phosphate-buffered saline (PBS) solution, fixed with 4% paraformaldehyde, and processed as previously described (Chang et al., 2014 (link)). Primary antibodies were used as follows: rabbit anti-GFP (Abcam; Cat# Ab6556; 1:1,000 dilution); mouse anti-VGAT (Synaptic Systems; Cat# 131 011; 1:1,000 dilution); guinea pig anti-VGLUT1 (Synaptic Systems; Cat# 135 304; 1:4,000 dilution). Secondary antibodies were used as follows: Alex Fluor 488 donkey anti-rabbit (Jackson Immuno Research; Cat# 711-545-152; 1:500 dilution); Rhodamine Red donkey anti-guinea pig (Jackson Immuno Research; Cat# 706-295-148; 1:500 dilution); Alexa Fluor 647 donkey anti-mouse (Jackson Immuno Research; Cat# 715-605-151; 1:500 dilution).
Images were captured on an inverted microscope with 60× objective (water, n/a 1.2) using a CCD camera (Princeton Instruments) and mercury lamp illumination under control of MetaMorph Software (Molecular Devices). Colocalization of background-subtracted images was analyzed in ImageJ using the Pearson’s coefficient (r) of the full images determined by the JACoP colocalization plug-in (Bolte and Cordelières, 2006 (link)).

Cells were cultured on cover-slips and transfected as above. Two days post transfection, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton-x100 in IF buffer (5% FCS, 2% BSA in PBS), then immunostained by primary anti-FLAG antibody (Sigma-Aldrich, F1804) followed by secondary AlexaFluor 647 Donkey Anti-Mouse (Jackson) antibody to image FLAG-tagged chaperones. The far-red secondary antibody was used to eliminate fluorescence overlap between the YFP/GFP-tagged aggregates and the chaperones. The cells were additionally stained with DAPI to mark nuclei.