This paper describes improvements in a method for detecting antigen in tissue cells using fluorescent antibody. The method involves conjugating antibody with fluorescein isocyanate, which allows for specific histochemical staining of antigen-antibody precipitates under a fluorescence microscope. The technique has been applied to detect antigens of rickettsiae, mumps virus, and pneumococcal capsular polysaccharide. The synthesis of fluorescein isocyanate involved separating and purifying two isomers of nitrofluorescein through fractional crystallization of their diacetates. The resulting fluorescein isocyanate was then used to label antibodies. The optimal conditions for conjugation were determined, with 0.05 mg of isocyanate per mg of protein yielding the best results. The choice of isomer was found to be less important, as both isomers stained tissues containing homologous antigen effectively. Tissue sections were prepared using a method that preserves antigenic activity and tissue architecture. The technique of staining involved applying the conjugate to tissue sections and examining them under a fluorescence microscope. The method was found to be effective for detecting antigens in various tissues, including those of monkeys infected with mumps virus. The use of fluorescence as a staining method offers advantages over other techniques, including good optical resolution and ease of use once the conjugates are prepared. However, non-specific staining can occur, which can be removed by shaking the conjugates with liver powder. Controls were used to ensure that observed staining was immunologically specific. The method has been successfully applied to detect pneumococcal polysaccharide, rickettsiae, and mumps virus. It is also likely applicable to other antigenic substances that can produce antibodies. The method is not ideal for examining unorganized materials unless the antigen is particulate and concentrated. However, it is well-suited for studying tissue sections, where the architecture allows for correlation of staining with location. The method has potential for use in the titration of viruses and rickettsiae due to its sensitivity.This paper describes improvements in a method for detecting antigen in tissue cells using fluorescent antibody. The method involves conjugating antibody with fluorescein isocyanate, which allows for specific histochemical staining of antigen-antibody precipitates under a fluorescence microscope. The technique has been applied to detect antigens of rickettsiae, mumps virus, and pneumococcal capsular polysaccharide. The synthesis of fluorescein isocyanate involved separating and purifying two isomers of nitrofluorescein through fractional crystallization of their diacetates. The resulting fluorescein isocyanate was then used to label antibodies. The optimal conditions for conjugation were determined, with 0.05 mg of isocyanate per mg of protein yielding the best results. The choice of isomer was found to be less important, as both isomers stained tissues containing homologous antigen effectively. Tissue sections were prepared using a method that preserves antigenic activity and tissue architecture. The technique of staining involved applying the conjugate to tissue sections and examining them under a fluorescence microscope. The method was found to be effective for detecting antigens in various tissues, including those of monkeys infected with mumps virus. The use of fluorescence as a staining method offers advantages over other techniques, including good optical resolution and ease of use once the conjugates are prepared. However, non-specific staining can occur, which can be removed by shaking the conjugates with liver powder. Controls were used to ensure that observed staining was immunologically specific. The method has been successfully applied to detect pneumococcal polysaccharide, rickettsiae, and mumps virus. It is also likely applicable to other antigenic substances that can produce antibodies. The method is not ideal for examining unorganized materials unless the antigen is particulate and concentrated. However, it is well-suited for studying tissue sections, where the architecture allows for correlation of staining with location. The method has potential for use in the titration of viruses and rickettsiae due to its sensitivity.