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Published on: December 16, 2013
Immunofluorescence staining of live lymph node tissue slices
Benjamin D Groff1, Andrew W L Kinman1, Jacob F Woodroof1
1Department of Chemistry, University of Virginia, Charlottesville, USA.
Insights
Researchers developed a new immunostaining method for live lymph node tissue slices, enabling detailed spatial mapping of immune cells while preserving tissue function. This technique allows visualization of immune cell organization and dynamics in a living environment.
Area of Science:
- Immunology
- Cell Biology
- Tissue Engineering
Background:
- Assessing immune system organization typically involves fixed tissue sections or specialized live-cell imaging techniques.
- Existing methods for live tissue analysis often rely on genetic reporters or pre-labeled cells, limiting flexibility.
- There is a need for methods that allow direct immunostaining and imaging of live, thick tissue explants.
Purpose of the Study:
- To validate a protocol for immunostaining and imaging live, thick lymph node tissue slices.
- To maintain the viability and functionality of lymph node slices during immunostaining.
- To provide a spatial map of immune cell organization within live lymph node tissue.
Main Methods:
- Developed and optimized an immunostaining protocol for live, thick lymph node slices.
- Tested staining parameters including time, temperature, and wash duration using anti-B220/CD45R antibodies.
- Verified antibody specificity using isotype controls and assessed staining depth and impact on cell function.
Main Results:
- Immunostaining signal was detectable up to 120 μm deep in live tissue slices.
- The protocol successfully revealed changes in B cell organization in lymph nodes from immunized mice.
- Most cell surface staining did not induce cytokine secretion, and T cell responses remained unaffected; however, anti-CD3 staining activated the tissue.
Conclusions:
- This novel method allows for spatial mapping of immune cells in live lymph node tissue while preserving cellular viability and function.
- The technique is effective for visualizing immune cell dynamics, such as T cell activation marker distribution.
- Careful selection of immunostaining reagents is crucial to avoid unintended cellular activation in live tissue.
Abstract:
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')2 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.
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