Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at -25 °C for Several Years

Int J Mol Sci. 2021 Aug 25;22(17):9180. doi: 10.3390/ijms22179180.


Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at -25 °C for several years (4-6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

Keywords: Japanese macaque monkey; corticotrophin-releasing factor; dense-cored neurosecretory vesicle; glutamate; paraventricular nucleus of the hypothalamus; post-embedding immunoelectron microscopy; vasopressin.

MeSH terms

  • Animals
  • Cryopreservation / methods*
  • Cryopreservation / standards
  • Female
  • Fixatives
  • Formaldehyde
  • Hypothalamo-Hypophyseal System / cytology*
  • Macaca fuscata
  • Male
  • Microscopy, Immunoelectron / methods
  • Microscopy, Immunoelectron / standards
  • Neurons / metabolism
  • Neurons / ultrastructure*
  • Tissue Fixation / methods*
  • Tissue Fixation / standards
  • Vasopressins / metabolism
  • Vesicular Glutamate Transport Proteins / metabolism


  • Fixatives
  • Vesicular Glutamate Transport Proteins
  • Vasopressins
  • Formaldehyde