Preparation of the Popliteal LN
For all the following steps, the mouse should be transferred to the heated platform (see Fig 1A). The use of this stage simplifies the management of the mouse during the preparation of the sample and contributes to the overall stability of the intravital setup.
1. Shave the hair of the left lower hind leg using a small animal clipper.
2. Tape the tail of the mouse to the right side of the body using surgical tape. The left lower hind leg is gently pulled out and kept in extension using surgical tape as shown in Fig. 1B.
3. Perform a skin incision on the area of the popliteal space and remove a small circular piece of skin.
4. Carefully expose the popliteal LN by removing some of the fat covering the popliteal cavity (see Note 3).
5. Once exposed, cover the LN with a small piece of moist gauze. Immobilization of the leg is then achieved by preparing a plaster cast.
6. Cut two rectangular pieces of plaster bandage (80 x 25 mm), quickly immerse them in water and drain off the excess water. Place the plaster bandages one on the top of the other with the left leg lying in the middle (see Fig. 1B). Plaster bandages are folded twice to create a block of plaster on each side of the leg (see Fig. 1C). The height of the plaster blocks should match that of the LN surface (see Note 4). The two plaster bandages are connected at the ankle level to complete immobilization of the leg (see Fig. 1D).
7. Allow the plaster cast to dry (see Note 5). The shape of the plaster cast is designed (1) to prevent movements of the leg and movements as a result of breathing by fixing the leg to the heated platform and (2) to create a flat surface around the LN to attach a cover slip (see below).
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