To determine whether an immune system is able to support T-cell-dependent affinity maturation one needs antigens that induce well-characterized immune responses. This chapter describes the response of the BALB/c mouse to hapten 2-phenyl-4-ethoxymethylene-oxazolone (phOx) coupled to carrier protein chicken serum albumin (CSA). Immunization with this hapten carrier complex will induce the formation of germinal centers (GCs), the microenvironment where the process of affinity maturation takes place. Immunohistological techniques to label germinal centers and examine the development of the GC structure during the primary immune response are described. The process of affinity maturation can be studied by the analysis of the mutational pattern in the V Ox1 L-chain in single GC B cells directly dissected from frozen tissue sections. The V Ox1 L-chain sequences are amplified by polymerase chain reaction (PCR), cloned, and sequenced. Methods are described to analyze V-gene sequences. Potential difficulties in the interpretation of the mutational pattern are discussed.
