Lab 22: Staining the B and T cell zones of the spleen using immunofluorescence
In this lab, the spleen is studied under immunofluorescent conditions, with the T and B cells being stained with different immunoflourescent dyes.
The spleen in humans is about the size of a fist and is a peripheral lymphoid organ. It collects antigen from blood and filters RBCs. In the spleen there are different zones and a distinct organization of the lymphoid tissue.
Red Pulp – bulk of the spleen, the site of red blood cell disposal
White Pulp – the area where the lymphocytes surrounding the arterioles entering the organ
PALS (Periarteriolar lymphoid sheath) – Where T cells are located and the inner region of the white pulp, and where lymphocytes and antigen-loaded dendritic cells come together in the periarteriolar lymphoid sheath.
Blood carries lymphocytes and antigen from a trabecular artery into a centrial arteriole. Cells and antigen then pass into a marginal sinus and drain into a trabecular vein. The marginal sinus is surrounded by a marginal zone of lymphocytes. The follicles consist of mainly B cells and the germinal center is surrounded by a B cell zone.
B cells and T cells are then activated when they encounter
antigen, by two signals. T cells are
activated when their specific antigen binds to the TCR. Once bound, an APC (such as a dendritic cell)
gives the second signal. B cells are
activated when the specific Ag is bound to its BCR. From there a T lymphocyte provide T cell help
to give the second signal. If the B cell
or T cell does not encounter its specific antigen, then it keeps circulating in
the blood until it does.
Spleen cells are frozen and then a cryo-section is preformed to obtain
razor-thin type spleen slices of a mouse.
From there it is stained in various dyes.
Procedure:
Follow handout.
Obtain slice of spleen and fixiate it on 95% ethanol.
Add Normal Goat Serum (NGS) which serves as a blocker for non-specific binding
Add the appropriate stains:
FITC – stains the anti-Ig found on B cells
PE – stains T cells
Both PE and FITC – stains both B cells and T Cells
Observations:
Discussion:
In this lab the structure, organization, and functionality of the spleen was studied. It was found that there is a distinct zone where B cells reside in (on the lower half) and where T cells reside (upper half). This was observed using immunofluorescence microscopy. FITC stained the T cells, while PE stained B cells. Depending on the filter used (green or red) each zone could be seen.
FITC stained the T cells green (when using the green cells). As observed in the upper half there is a bright, slightly dispersed area. PE stained the B cells red (depending on the filter used) and this was seen in the lower half. It was important not to confuse the B cells seen with the RBCs present outside the cell. This was done by recalling that the white pulp separates the two regions of B cells and RBCs.