Immunophenotyping of Spleen and LN Cells

Spleens and popliteal LNs were harvested at indicated times and prepared as previously described^{57 (link)}. Briefly, tissues were fixed in periodate-lysine-paraformaldehyde buffer and placed in 30% sucrose in PBS. Tissues were then embedded in OCT medium (Electron Microscopy Sciences), frozen in dry-ice cooled isopentane and sections were cut on a cryostat (Leica Microsystems). Sections were blocked in 5% sera and stained with the following antibodies to: CD11b (M1/70, eBioscience), CD11c (N418, eBioscience), B220 (RA3–6B2, eBioscience), CD64 (polyclonal, R&D Systems), MHC II (I-A/I-E, M5/114.15.2, eBioscience), followed by the relevant secondary antibodies conjugated to fluorophores. Images were acquired using Leica SP8 microscope and analyzed using Imaris software (Bitplane).

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Baharom F., Ramirez-Valdez R.A., Tobin K.K., Yamane H., Dutertre C.A., Khalilnezhad A., Reynoso G.V., Coble V.L., Lynn G.M., Mulè M.P., Martins A.J., Finnigan J.P., Zhang X.M., Hamerman J.A., Bhardwaj N., Tsang J.S., Hickman H.D., Ginhoux F., Ishizuka A.S, & Seder R.A. (2020). Intravenous Nanoparticle Vaccination Generates Stem-Like TCF1+ Neoantigen-Specific CD8+ T Cells. Nature immunology, 22(1), 41-52.

Publication 2020

OCT medium cryostat CD11b (M1/70, CD11c (N418, B220 (RA3–6B2, CD64 (polyclonal,

Antibodies Buffer Cd11b Dry ice Electron microscopy Frozen Isopentane Microscope Periodate lysine paraformaldehyde Sera Sucrose Tissues

Corresponding Organization :

Other organizations : National Institutes of Health, National Institute of Allergy and Infectious Diseases, Agency for Science, Technology and Research, Singapore Immunology Network, Avidea Technologies (United States), Icahn School of Medicine at Mount Sinai, SingHealth

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Variable analysis

independent variables

Harvesting time points for spleens and popliteal lymph nodes (LNs)

dependent variables

Cellular phenotypes observed through immunostaining and microscopy analysis

control variables

Tissue fixation method (periodate-lysine-paraformaldehyde buffer)
Tissue processing (30% sucrose in PBS, embedding in OCT medium, freezing in dry-ice cooled isopentane)
Sectioning method (cryostat)
Blocking and antibody staining procedure
Imaging platform (Leica SP8 microscope) and analysis software (Imaris)

controls

No positive controls specified
No negative controls specified

Annotations

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