A technique for decalcification and demonstration of substance P-like immunoreactivity

Substance P-like immunoreactivity (SPLI) was demonstrated in mouse spinal cord by an indirect immunofluorescence method after decalcification of the vertebra with a mixture of EDTA and Zamboni's fixative. SPLI was observed mainly in the gray matter of the spinal cord, especially the superficial layers of the dorsal horn; the distribution was the same as in the control spinal cord. No diffusion and depletion of SPLI were recognized after decalcification and no specific fluorescence was observed. The findings reported here indicate that decalcification with a mixture of EDTA and Zamboni's fixative is a useful method for examining SPLI in nervous tissue surrounded in situ by calcified tissues.