3.1 Immunostaining for mycobacterial infections using BCG antiserum

Indirect immunoperoxidase staining using BCG rabbit antiserum (Dako/Agilent Technologies) diluted at a 1:5000 was highly sensitive for detecting mycobacteria in histopathological sections [4, 7, 8, 24, 25]. No pretreatment for antigen retrieval was given. Mycobacterial antigens were clearly demonstrable not only in caseous necrosis in active tuberculosis but also in a fibrous nodule of old calcified tuberculosis (Figure 1). BCG antigens were scarcely detectable in epithelioid granulomas. The BCG immunostaining was much more sensitive to detect mycobacteria than conventional acid-fast (Ziehl-Neelsen) staining, as indicated in Table 2 [4, 24]. In BCG immunostaining, the judgment of positivity can be done easily and quickly, while it takes minutes or longer in case of conventional acid-fast staining. It is evident that the antiserum is reactive with mycobacterial antigenic substances on destroyed bacterial fragments. The BCG immunostaining was also applicable to demonstrating non-tuberculous mycobacteria and Mycobacterium leprae. Figure 2 illustrates positive findings in opportunistic Mycobacterium avium-intracellulare (MAI) infection in AIDS and in lepromatous (multibacillary) leprosy in a biopsied skin lesion. No positivity was seen in tuberculoid (paucibacillary) leprosy.

The BCG antigens were extremely stable after prolonged fixation in formalin for a long period of time [19, 26]. Figure 3 displays dense-positive signals in an exudative pulmonary tuberculosis lesion fixed in formalin for nearly 70 years.
