A tetramethylbenzidine/tungstate reaction for horseradish peroxidase histochemistry

Tracing of neuroanatomical pathways commonly involves the histochemical demonstration of horseradish peroxidase, using the chromogen tetramethylbenzidine. A new modification of this reaction using ammonium paratungstate stabilizer retains high sensitivity while permitting the reaction to be performed at pH 6.0 in isotonic solutions. The reaction product resists solvents, allowing Nissl-stained sections to retain their peroxidase labeling. With subsequent stabilization by diaminobenzidine, the tissue is suitable for electron microscopic study and is compatible with post-embedding immunocytochemistry.     

Expression of the major surfactant-associated protein, SP-A, in type II cells of human lung before 20 weeks of gestation

In a previous paper (Otto-Verberne et al., Anat. Embryol. 178, 29-39 (1988) we reported that the type II alveolar epithelial cell can be identified in fetal human lung on the basis of morphological and immunological characteristics from 10 to 12 weeks after conception (a.c.) onward. For immunological recognition we used a lung-specific antibody, called SALS-Hu (specific anti-lavage serum, rabbit antihuman). The present immunoblotting experiments, after one-and two-dimensional electrophoresis, showed that SALS-Hu-reactive proteins in lavage fractions obtained from alveolar proteinosis patients exhibited molecular masses of mainly 29, 31 to 36, and 62 to 66 kDa. All SALS-Hu-reactive proteins migrated in the same acidic isoelectric point range (pI 4.4-5.1) and were almost undetectable when we used SALS-Hu preabsorbed with recombinant surfactant-associated protein A. We concluded that SALS-Hu recognizes exclusively isoforms of the major surfactant-associated protein, SP-A. In vitro translation assays in which we used mRNA isolated from adult human lung confirmed that SALS-Hu recognized the 29 to 31 kDa SP-A precursor proteins. These SALS-Hu-immunoreactive precursors for SP-A were already detectable (though in much lower amounts) in human fetuses aged 17 to 18 weeks, indicating that mRNA coding for SP-A is present at that time. We concluded that the cytoplasmic staining of fetal (from 10-12 weeks a.c. onward) and adult human type II cells by SALS-Hu is due to the presence of SP-A.