Figure 4.

Trabecular sinus, perifollicular sinus and blood vessels interconnections in relation with the perifollicular macrophages. (A) Thick slice of tracheobronchial LN were cleared using iDISCO+ and (B–D) different area and magnifications of cleared LN slice stained with anti-CD169 and anti-CD31 antibodies. Imaged with multiphoton microscope A1RMP+ with an objective lens 25x and dual excitation at 960 nm and 1040nm. From 960 nm excitation, Second Harmonic generation (SHG) was acquired in blue channel in forward direction and fluorescence of Alexa Fluor 488 was acquired in green channel in backward direction. From 1040 nm excitation, fluorescence of Alexa Fluor 555 was acquired in red channel in backward direction. F, follicle; Tb, Trabeculae. Red arrows: T cell area blood endothelial cells, pink arrows: peri-trabecular lymphatic endothelial cells, dark blue arrows: perifollicular lymphatic endothelial cells, green arrows: perifollicular macrophages, orange arrows: lymphatic-blood capillaries connections. (A–C), scale bar 100µm.