Alexa fluor 488 anti cd8a

Orthotopic allografts were generated as in the efficacy studies. When tumors reached 200–400 mm³ (about 2 weeks post-cell injection), R848-Toco/HA-Toco (25 μg on R848 basis) in 50 μL volume was injected intratumorally. After 24 h, tumors were dissected and embedded in OCT medium (Fisher Scientific), followed by storage at −80 °C. Tumors were sectioned (10 μm) with a cryotome. Sections were fixed for 2 × 10 min with acetone and washed with PBS. Primary antibodies were diluted to 5 μg/mL in blocking buffer (5% goat serum in PBS) and incubated overnight at 4 °C. Antibodies used were Alexa Fluor® 488 anti-CD8a, Alexa Fluor® 594 anti-CD11b, and Alexa Fluor® 647 anti-CD11c (BioLegend). After antibody staining, sections were stained with DAPI (0.5 μg/mL in PBS; Invitrogen) for 10 minutes, mounted in SouthernBiotech™ Fluoromount-G™ Slide Mounting Medium and stored in the dark at 4 °C. Images were acquired using an Olympus IX-81 inverted epifluorescence microscope at 10x magnification. and images were captured with a Hamamatsu EM-CDD Digital Camera. The entire tumor section was imaged. Many 10x images were montaged together using SlideBook 6 to view the whole section.

Cis-Dichlorodiamine platinum (II) (cisplatin) was purchased from Strem Chemicals, Inc. (Newburyport, MA). Sodium hyaluronate was purchased from Contipro Inc (Dolní Dobrouč, Czech Republic). 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), and 3-(3-Dimethylaminopropyl)-1-ethyl-carbodiimide hydrochloride (EDC-HCl) was purchased from Chem-Impex International, Inc. (Wood Dale, IL). 1-Hydroxybenzotriazole hydrate (HOBt•H₂O) was purchased from Advanced ChemTech (Louisville, KY). N-(tert-Butoxycarbonyl)glycine (Boc-Gly-OH), hydrogen peroxide solution (H₂O₂, 30 wt.% in H₂O), succinic anhydride, (±)-α-tocopherol, N,N′-Dicyclohexylcarbodiimide (DCC), N,N-Dimethylpyridin-4-amine (DMAP), trifluoroacetic acid (TFA), N,N-Diisopropylethylamine (DIPEA), oxaliplatin, tetrabutylammonium hydroxide solution (TBA-OH, 40 wt.% in H₂O), and anhydrous organic solvents were purchased from Sigma-Aldrich (Saint Louis, MO). Alexa Fluor ® 488 anti-CD8a, Alexa Fluor® 594 anti-CD11b, and Alexa Fluor® 647 anti-CD11c antibodies were purchased from BioLegend (San Diego, CA). All other chemicals and organic solvents were purchased from Thermo Fisher Scientific (Waltham, MA).