Camera software for dp80

Fixed-tumor tissues were excised from tumor-bearing mice and fixed using IHC Zinc Fixative (BD Bioscience, San Jose, CA, USA). Five micrometer paraffin-embedded sections were prepared for Immunohistochemical staining with Tyramide Signal Amplification (Opal Multiplex IHC Assay, PerkinElmer, Waltham, MA, USA) to examine the percentage of TILs. The following primary antibodies were used: rat anti-mouse CD3 (CD3-12), rabbit anti-mouse CD4 (EPR19514), rat anti-mouse CD8a (4SM15), rat anti-mouse Foxp3 (FJK-16S), rabbit anti-mouse PD1 (EPR20665). The number of immune cells per section was recorded blind to genotype and normalized to core area. Individual core counts from 10 or more replicates were available for most cases, and immune cell counts per square millimeter were averaged across replicates. Cut-off values of low versus high immune cells were defined by the midpoint. Slides were examined with an Olympus BX51 microscope using Camera Software for DP80 (Olympus America Inc., Center Valley, PA, USA). The cell count of TILs was determined using ImageJ. The density of TILs calculation as follows: $density of TIL=\frac{cells count number}{size of image field of view (m{m}^2)}$
$\begin{array}{ll}image field of& view \left(height, width, diagonal\right)\\ & =\frac{\mathrm{CCD}\mathrm{sensor}\mathrm{size}\left(\mathrm{height},\mathrm{width},\mathrm{diagonal}\right)}{\mathrm{objective}\mathrm{magnification}\mathrm{x}\mathrm{adapter}\mathrm{magnification}}\end{array}$