Donkey anti rat alexa flour 647

B16F10 tumors 7d post intradermal implantation in the dorsal skin with 0.5 × 10⁶ B16F10 cells were surgically excised, embedded in optimum cutting temperature embedding medium, and stored at −80°C. A cryostat was used to slice 8 μm thick tissue sections that were mounted onto histological slides and stored at −20°C. Sections were 2% PFA fixed for 20 min at room temperature, blocked with 10% donkey serum diluted in Dulbecco’s Phosphate Buffered Saline (D-PBS) with calcium and magnesium for 1h at room tempertature, and incubated overnight at 4°C with the following primary antibody: goat anti-mouse CD62P (1:13, R&D Systems, AF737), and rat anti-mouse CD3 (1:50, Invitrogen) or rat anti-mouse CD31 (1:50, Invitrogen). The following day, the slides were incubated for 1h at room temperature with the following secondary antibody: donkey anti-goat Alexa Flour 555 (1:200, Invitrogen) and donkey anti-rat Alexa Flour 647 (1:1000, Invitrogen). In between each staining step, slides were washed three times with gentle agitation in 0.1% Tween 20 diluted in D-PBS with calcium and magnesium. Washed slides were mounted using Vectashied mounting medium with DAPI and microscopic images were taken using a Zeiss AxioObserver Z1 fluorescent microscope with a 10x magnification objective.