SMI-32 Staining Research Articles

Microwave-stimulated antigen retrieval is pH and temperature dependent.

The effects of the pH of solutions used in microwave-stimulated antigen retrieval were investigated for two antibodies commonly used in neurosciences, MAP-2 and SMI-32. A prolonged fixation time (longer than 1 week) prevents good immunohistochemical staining of these and many other antigens in paraffin and vibratome sections. On human brain tissue stored for 17 months in formaldehyde, the effects of different solutions during microwave treatment were compared, i.e., 4% aluminum chloride (pH 2.75), 0.1 N hydrochloric acid (pH 0.98), and citrate buffers (pH 2.5, 4.5, 6.0), together with the influence of temperature and microwave irradiation. We conclude that the pH and temperature of the solutions and the irradiation time are of great importance for the retrieval of antigens, but in a different way for the two antibodies studied. The SMI-32 staining results are optimal with citrate buffer at pH 2.5 and 2-hr irradiation at 90 degrees C, whereas MAP-2 staining results are optimal with citrate buffer at pH 4.5 and 10-min full-power irradiation. In the other conditions the staining is less intensive or less homogeneously distributed over the section.

Developmental changes in the heavy subunit of neurofilaments in the corpus callosum of the cat

In the corpus callosum of the cat, the heavy subunit of neurofilaments (NFH) can be demonstrated with the monoclonal antibody NE14, as early as P11, not at P3, and only in a few axons. At P18–19 and more markedly at P29, many more callosal axons have become positive to NE14 and this is similar to what is found in the adult. In contrast, callosal axons become positive to the neurofilament antibody SMI-32 only between P29 and P39 and remain positive in the adult. Treatment with alkaline phosphatase prevents axonal staining with NE14, but results in SMI-32 staining of a few callosal axons as early as P11, but not at P3. Between P11 and P19 the number of axons stained with SMI-32 after alkaline phosphatase treatment increases, in parallel with that of axons stained with NE14. Thus NE14 appears to recognize a phosphorylated form of NFH, while SMI-32 appears to recognize an epitope of NFH which is either masked by phosphate or inaccessible until between P29 and P39, unless the tissue is treated with alkaline phosphatase. These two forms of NFH appear towards the end of the period of massive developmental elimination of callosal axons 1. They are also synchronous with changes in the spacing of neurofilaments quantified in a separate ultrastructural study 3. These cytoskeletal changes may terminate the juvenile-labile state of callosal axons and allow further axial growth of the axon.