Immunofluorescence staining
tumors, Tumor
SECONDARY, TUMORS, TUMOR
Mouse tumors were collected and fixed in 10% formalin, embedded and sectioned (5 μm). Tumor tissue FFPE slides were then deparaffinized and rehydrated. For antigen retrieval, slides were maintained in antigen unmasking solution (Vector Laboratories; H-3300) at a subboiling temperature for 20 min using a microwave. Slides were washed three times for 5 min each in PBS supplemented with 0.1% Tween 20 and then were blocked with 5% BSA (Thermo Fisher Scientific) and 5% goat serum (Sigma-Aldrich) for 1 h at room temperature. The slides were incubated with anti-CD3 (Abcam; ab11089; 1:800 dilution) and anti-CD8 (Cell Signaling; 98941 S; 1:400 dilution) primary antibodies overnight at 4 °C. The next day, slides were washed as above and incubated with conjugated secondary antibodies (Thermo Fisher Scientific; A-11036 and A-11006) for 1 h at room temperature. The slides were then washed, stained for DAPI (Invitrogen) and mounted with SlowFade Diamond Antifade Mountant (Invitrogen). Once finished, the slides were scanned using a ZEISS Axio Scan slide scanner and analyzed using Halo software (Indica Labs). Image annotations were performed in a blinded manner. Cells stained with an intensity exceeding the settings threshold were counted as positive. The settings were set to include the full range of staining intensity (weak to strong). Halo counted the CD3