Chromogen 3 amino 9 ethylcarbazole

For immunohistochemical analysis, the PET/CT imaging and biodistribution cohort (n=6) tumors were used. Tumor ipsilateral right brain and contralateral left-brain samples were fixed in neutral phosphate-buffered 10% formalin (Fisher Scientific, Pittsburgh, PA) for 24 h. Samples were then transferred to 70% ethanol until the Zr-89 decayed (>5 half-lives). Samples were dehydrated using an EtOH gradient of 70% to 100% over 24 h, cleared with histology grade xylene (Fisher Scientific), embedded in granular paraffin, and sectioned (5 μm) on poly-lysine slides. Sections were incubated with 10% normal goat serum, and then overnight at 4°C with primary antibody. Biotinylated secondary goat anti-rabbit Ab (Abcam; dilution 1:200) was used to detect the primary Ab. The staining signal was amplified with avidin-biotin complex (Vector Laboratories, Burlingame, CA) and developed using chromogen 3-amino-9-ethylcarbazole (Sigma-Aldrich). ImageJ software (ImmunoRatio plugin) was used to quantify CD11b⁺ cells per nuclear area [20 (link)]. A histomorphologic analysis of the tumors was performed with the H&E slides.

ELISpot assays were carried out on Multiscreen HTS IP 0.45μm filter plates (Millipore Sigma). Briefly, the plates were coated with anti-IFNγ capture antibody (clone AN18, BioLegend) overnight at 4°C and blocked with RPMI-1640 supplemented with 20% FBS for 2 hours at 37°C. 500,000 splenocytes were then added, and the plates were incubated in the presence of 10 μg/ml of pooled peptides or 25 ng/ml of PMA and 0.5 μg/ml of Ionomycin in positive control wells, or 0.1% DMSO in negative control wells. In addition, for freeze-thawed splenocytes (Figure 7) 50 IU/ml of mouse IL-2 was added to the stimulation medium to enhance T cell survival. After incubating the plate for 44 hours at 37°C, the wells were washed to remove the cells. The wells were then incubated with biotinylated anti-IFNγ detection antibody (clone R4–6A2, BioLegend) for 2 hours at 37°C followed by streptavidin-HRP for 1 hour at room temperature. IFNγ spots were developed using 3-Amino-9-ethylcarbazole chromogen (Sigma) and images were captured by the ELISpot Reader (AID GmbH, Germany). Automatic spot counts were obtained using the AID ELISpot Reader software.