Donkey anti rat alexa 647

Harvested B16 tumors were fixed overnight in PLP fixative, followed by incubation in 15% and 30% sucrose. Tumors were embedded in OCT and frozen. Cryosections were cut at 5 μm thickness and stored at −20°C. Slides were stained with rat anti-mouse CD8 (clone CT-CD8a, Fisher) and donkey anti-rat Alexa 647 (Jackson Immuno Research Labs). Slides were washed and mounted in ProLong antifade reagent with DAPI (Life Technologies). Images were acquired using an AxioObserver D1 equipped with an X-Cite 120LED System.

Mice were infected with VACV in the center of each ear pinnae using a bifurcated needle. Five days post-infection, ears were harvested and embedded in Tissue-Tek OCT (Sakura Finetek), rapidly frozen by immersion in liquid nitrogen-cooled 2-methyl butane, and stored at -80°C. Lateral cryostat sections (10 μm) were cut at -20°C, mounted on glass slides and fixed for 30 min in 4% paraformaldehyde in PBS (pH 7.4). For both assays, three-dimensional images were collected on an Olympus IX81 fluorescent microscope, deconvolved and analyzed using Slidebook 6.0 software (Intelligent Imaging Innovations).
For RNA fluorescence in situ hybridization, mice were infected with VACV-OVA. To visualize plasma membranes, tissue was stained with Wheat Germ Agglutinin conjugated to CF488 (1:1000, Biotium). Tissue was fixed again and RNA was visualized using the ViewRNA Cell Plus Assay (Invitrogen) with probes recognizing murine IFN-β and gallus ovalbumin (Affymetrix) according to manufacturer’s specifications with the omission of using a protease. Nuclei were stained with DAPI.
For Immunofluorescence microscopy, mice were infected with VACV-NP-S-eGFP, VACV-CCL4, or VACV-mCherry. Tissue was stained with antibodies to Ly6C (clone HK1.4 1:2500, Abcam) and visualized using secondary donkey anti-rat Alexa 647 (1:500, Jackson Immunoresearch) or donkey anti-rat BV421 (1:100, Jackson Immunoresearch).