Fitc anti human mouse granzyme b antibody

Murine organs were fixed in phosphate-buffered 4% formaldehyde and embedded in paraffin. Three to five micrometres thick sections were stained with hematoxylin and eosin.
Immunofluorescence staining on 5-μm paraffin sections using antibodies against cleaved caspase3 (Cell Signaling, #9664), Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 (ThermoFisher, #A-11011), and FITC anti-human/mouse Granzyme B Antibody (BioLegend, #515403) was performed as described^{8 (link),51 (link),52 (link)}.

Cells in single-cell suspension were fixed with 2% PFA (Santa-Cruz, #sc-281692). After washing with a Flow Cytometry Staining Buffer (eBioscience, #00–4222–26), cells were stained with antibodies for cell-surface marker for 1 h on ice in the dark. For staining of intracellular proteins, the cells were washed and resuspended in the permeabilization buffer (BD, #554723) and stained by antibodies in the permeabilization buffer for 1 h on ice in the dark. The cells were then pelleted and resuspended in the Flow Cytometry Staining Buffer for flow cytometry analysis. Antibodies used for flow cytometry include: V450 Mouse Anti-Mouse CD45.2 (BD Horizon, #560697, Clone 104), APC anti-mouse NK1.1 Antibody (BioLegend, #108710, clone PK136), FITC anti-human/mouse Granzyme B Antibody (BioLegend, #515403, clone GB11), CD8a Monoclonal Antibody (53–6.7) PE-Cyanine7 (eBioscience, #25–0081–82, clone 53–6.7), CD4 Monoclonal Antibody (RM4–5), and PE (eBioscience, #12–0042–82, clone RM4–5).

Rabbit anti-cystatin F polyclonal antibody, recombinant cistatin F and mouse 1D10 anti-cathepsin H Mab was prepared by our group in Ljubljana as reported [45 (link), 50 (link)]. Rabbit N1 antibody against N-terminal of cystatin and Δ15N cystatin F were prepared at Dundee University, Dundee, UK, [54 (link)]. Goat anti-cathepsin C pAb was from R&D Systems (Minneapolis, MN), mouse anti-GAPDH mAb was from Invitrogen and rabbit anti-CRK-L pAb was from Sigma. The dye identifying the lysosomes, LysoTracker, was from Molecular Probes (Eugene, OR). Secondary labelled antibodies goat anti-rabbit Alexa Fluor 488 and donkey anti-goat Alexa Fluor 555 were from Molecular Probes (Eugene, OR). Antibodies to CD16 were purchased from Biolegend (San Diego, CA). FITC anti-human/mouse granzyme B antibody was purchased from Biolegend (San Diego, CA).