Edta ph 9

Murine breast cancer tumors obtained through in vivo experiment described above. Samples were stained for CCT2 using anti-CCTβ antibody (LS-B4861; LifeSpan Biosciences) Antibodies were diluted 1:100 in Antibody Diluent (Leica). Staining of tissue arrays was performed using a Bond-Max Immunostainer (Leica), with an epitope retrieval buffer of EDTA pH 9.0 (Leica). Polymer Refine Detection reagents (Leica) were used, which include a hematoxylin counterstain. Scoring of staining was performed by a surgical pathologist as previously published^{14 (link),16 (link)}.

Specimens from necropsied mice were fixed in 10% neutral buffered formalin (Surgipath Leica, Buffalo Grove, IL, USA) and embedded in paraffin. Five-micron sections were sliced using a rotary microtome (Leica). SelecTech hematoxylin and eosin reagents (Surgipath, Buffalo Grove, IL, USA) were used for histological staining. For immunohistochemistry, antigen retrieval was performed with either sodium citrate, pH 6.0, or EDTA, pH 9.0 (Leica, Buffalo Grove, IL, USA). Primary antibodies were against MYC (Abcam, Cambridge, MA, USA), MPO (Thermo Fisher Scientific, Waltham, MA, USA) or F4/80, CD86, CD3, CD4, or CD8 (all from Cell Signaling Technology, Danvers, MA, USA). A Bond-Max Immunostainer and Polymer Refine Detection reagents (Leica, Buffalo Grove, IL, USA) were used for staining. Multiple fields of view of each tumor section were imaged across three representative mice from each treatment group on a Keyence BZ-X800 microscope. Antibody detection was quantified using Keyence BZ-X800 analysis software. Results are reported as mean ± SD. To compare each mean with every other mean, a one-way ANOVA followed by post hoc Tukey’s test for multiple comparison was used to analyze statistical significance between treatment groups. (p < 0.05 [*], p < 0.01 [**], p < 0.001 [***], p < 0.0001 [****]).