Tracing T Cell Trafficking in Lungs

RAG2-deficient mice were injected intravenously with 0.1 × 10⁶ KPP-eGFP cells and 4 × 10⁶ tdTomato⁺ CD3⁺ T cells isolated as described below. Then, 8 days after intravenous injection, the mice were euthanized, and 20 ml of cold PBS/heparin 5 IU ml⁻¹ solution was perfused directly into the right ventricle using a 27-gauge needle. Lungs were isolated by dissection and tissues were fixed using 4% paraformaldehyde in PBS^{23 (link)}. Tissue clearing was performed using the FluoClearBABB approach^{27 (link)} and whole-mount images were then acquired using a SP8 microscope equipped with a white-light laser and the HCX APO L ×20/0.95 NA IMM lens (Leica Microsystems). Imaging data were analysed on a workstation (Thinkmate) using Imaris software (Bitplane).