The co-existence of catecholamines with opioid peptides in adult hamster adrenal chromaffin cells was studied by Sternberger's peroxidase-antiperoxidase (PAP) method. Paraffin embedded serial sections (3-4μm thick) of adrenal glands perfusion-fixed with either Bouin's fluid or 4% paraformaldehyde solution were immunostained using anti-adrenaline (AP16-2), -noradrenaline (NAP1-18), -Met-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-Enk-Arg-Gly-Leu: R-0171, NE2-206) and -Met-enkephalin-Arg⁶-Phe⁷ (Met-Enk-Arg-Phe: AP3-311) sera.
1. Selective and specific immunostaining for adrenaline and noradrenaline was obtained in tissues fixed in 4% paraformaldehyde solution. Bouin's fluid gave negative or unsatisfactory results.
2. Immunostaining with two kinds of Met-Enk-Arg-Gly-Leu (R-0171, NE2-206) and one kind of Met-Enk-Arg-Phe sera (AP3-311) was dependent upon the method of fixation used. The immunostaining in the Bouin-fixed tissues was stronger than that in the 4% paraformaldehyde-fixed ones.
3. Secretory granules in chromaffin cells were responsible for both adrenaline- and noradrenaline-like and Met-Enk-Arg-Gly-Leu- and Met-Enk-Arg-Phe-like immunoreactivities.
4. All adrenaline-storing (A) and noradrenaline-storing (NA) cells showed intense Met-Enk-Arg-Gly-Leu- and Met-Enk-Arg-Phe-like immunoreactivities. In the Bouin-fixed adrenal medulla, immunostaining of A cells with the two kinds of anti-Met-Enk-Arg-Gly-Leu sera (R-0171, NE2-206) was deeper than that of the NA cells. Immunostaining of A cells with the anti-Met-Enk-Arg-Phe serum (AP3-311) was apparently of the same intensity as that of NA cells. In 4% paraformaldehyde-fixed tissues, no correlation was seen between the distribution of A and NA cells and that of intensely and slightly Met-Enk-Arg-Gly-Leu and Met-Enk-Arg-Phe immunopositive cells.
The “masking” of immunoreactive residues in chromaffin granules by an aldehyde-noradrenaline complex may be one of the major causes of the decreased opioid peptide-like immunoreactivity in the 4% paraformaldehyde-fixed adrenal medulla.