granzyme b antibody  (Bio-Techne corporation)

Journal: bioRxiv

Article Title: Unbiased metastatic niche-labeling identifies estrogen receptor-positive macrophages as a barrier of T cell infiltration during bone colonization

doi: 10.1101/2024.05.07.593016

Figure Lengend Snippet: (A) CD8 T cell quantification was conducted in the hindlimb bones of mice injected with LLC1 cells. This included Esr1 fl/fl mice with and without anti-PD1 treatment (n=5 mice each), as well as LysM-Cre Esr1 fl/fl mice (n=5 mice). The frequency of CD8 T cells remained unaffected by Esr1 knockout; however, it significantly increased following anti-PD1 treatment. (B) A representative series of immunofluorescence images illustrates the heightened infiltration of CD8 T cells into the tumor region in bone metastatic tumors of LysM-Cre Esr1 fl/fl mice compared to control mice. In the images, CD8 T cells are marked in white, F4/80+ macrophages in red, and LLC1 tumor cells in green. (C) Quantification of tumor-infiltrating CD8 T cells in LysM-Cre Esr1 fl/fl mice compared to their control Esr1 fl/fl counterparts. (D) A representative series of immunofluorescence images illustrates the enhanced infiltration of Granzyme B+ CD8 T cells into the tumor region in bone metastatic tumors of LysM-Cre Esr1 fl/fl mice compared to control mice. In the images, CD8 T cells are marked in red, Granzyme B in white, and LLC1 tumor cells in green. (E) Quantification of tumor-infiltrating Granzyme B+ CD8 T cells in LysM-Cre Esr1 fl/fl mice compared to their control Esr1 fl/fl counterparts. (F) Quantification of biotin+ CD8 T cells was carried out in the hindlimb bones of mice treated with either fulvestrant or anti-PD1 following IIA injection with SAMENT+ LLC1 cells. The findings suggest that compared to anti-PD1 treatment, fulvestrant enhances the infiltration of CD8 T cells into metastatic tumors. Control group: n=5 mice; Fulvestrant group: n=5 mice; anti-PD1 group: n=5 mice. (G) A series of representative immunofluorescence images vividly depict the heightened infiltration of CD8 T cells into the tumor region within bone metastatic tumors of mice subjected to fulvestrant treatment. In these images, CD8 T cells are highlighted in white, F4/80+ macrophages in red, and LLC1 tumor cells in green. The infiltrated CD8 T cells are indicated by yellow arrows. (H) Cancer cell quantification was performed in the hindlimb bones of mice injected with LLC1 cells, including LysM-Cre Esr1 fl/fl mice (n=5 mice) and their control Esr1 fl/fl counterparts (n=5 mice), with or without anti-PD1 treatment, following IIA injection. Data are presented as geometric mean ± geometric SD in A, C; mean ± SEM in E, F, H. P values were assessed by repeat measure one-way ANOVA followed by least significant difference (LSD) test in A; by unpaired t-test in C, E, F; by Fisher least significant difference test post repeat measure two-way ANOVA test in H.

Article Snippet: Primary antibodies used in this study were as follows: anti-Biotin-APC(Miltenyi Biotec,130-113-288), anti-mRFP(Rockland,600-401-379), anti-GFP(Novus Biologicals,NB100-1614), anti-Granzyme B(Novus Biologicals,NB100-684), anti-F4/80(Cell Signaling technology,70076,for paraffin section), anti-F4/80(BIO-RAD,MCA497,for frozen section), anti-ERa(Thermo Fisher Scientific,MA1-80216), anti-CD8a(Thermo Fisher Scientific,14-0195-82), anti-ly6G(BioLegend,127602), anti-human CD68(Proteintech,25747-1-AP), anti-human Cytokeratin 8(DSHB,TROMA-I) and anti-human Cytokeratin 19(DSHB,TROMA-III).

Techniques: Injection, Knock-Out, Immunofluorescence, Control