Immunofluorescence staining of live lymph node tissue slices.

Explants of lymphoid tissue provide a rare opportunity to assess the organization of the immune system in a living, dynamic environment. Traditionally, ex vivo immunostaining is conducted in fixed tissue sections, while live tissues are analyzed using genetically engineered fluorescent reporters or adoptively transferred, pre-labelled cell populations. Here, we validated a protocol for immunostaining and imaging in live, thick slices of lymph node tissue, thus providing a spatial "map" of the lymph node while maintaining the viability and functionality of the slices. Using anti-B220/CD45R (B cell) as a prototype antibody, the procedure for immunostaining was tested for sufficient signal to noise with respect to staining time, temperature, and wash time, and the specificity was verified in comparison to isotype controls. Immunostaining signal in live tissue slices was detectable to atleast 120 μm deep for both whole antibodies and F(ab') fragments using the staining procedure. This procedure revealed the expected changes in B cell organization in lymph nodes from immunized mice. Cell surface staining with most antibodies did not induce cytokine secretion, and cytokine secretion in response to T cell stimulation was unaffected by immunostaining. Staining with known a mitogenic antibody (anti-CD3) simultaneously labelled the cells and activated the tissue, confirming that reagents for live immunostaining must be selected judiciously. As a proof of concept, this method was used to reveal the dynamic distribution of CD69, a T cell activation marker, in lymph node slices before and after ex vivo stimulation.