Potassium ferric ferrocyanide

Ten micron – thick frozen sections of the tumors were washed and rehydrated in TBS prior to permeabilization in 0.3% Triton X-100 in TBS for 15 min. Protein block was performed using Super Block (Scytek, Logan, UT) for 7 min. The sections were then blocked for endogenous peroxidase activity for 5 min in 0.3% H₂O₂ solution in TBS. After 2 washes in TBS, rat primary antibody against F4/80 (Abcam, Cambridge, MA) was diluted to 5 μg/ml in TBS, and incubated for 90 min at room temperature. Primary antibody was detected using horseradish peroxidase conjugated goat-anti-rat secondary antibody (Vector Laboratories, Burlingame, CA) after 30 min incubation at room temperature. The slides were then incubated with activated diaminobenzidine (DAB) solution for 5 min. After DAB, slides were PB stained with 2% potassium ferric-ferrocyanide (Sigma, St. Louis, MO) in 3.7% hydrochloric acid, and then counterstained with Nuclear Fast Red (Sigma). The slides were then dehydrated, and scanned using Aperio Scanscope CS (Leica Biosystems).

Labeled cells were fixed and 10⁵ cells were prepared on a cytospin slide. The slide was PB stained with 10% potassium ferric-ferrocyanide (Sigma, St. Louis, MO) in 3.7% hydrochloric acid and counterstained with Nuclear Fast Red (Sigma) and scanned using Aperio Scanscope CS (Leica Biosystems). Human MSC and NSC incubated with various combinations of HPF or FHP and stained with PB alone or with 3,3'-diaminobenzidine enhancement revealed approximately 100% of cells were labeled.