Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology

doi:10.1016/0196-9781(86)90076-8

Peptides

Volume 7, Issue 1, January–February 1986, Pages 155-159

https://doi.org/10.1016/0196-9781(86)90076-8 Get rights and content

Abstract

Use of an ethylene glycol based cryoprotectant solution has been found to be effective for the long-term storage of brain tissue either in block form or as freely floating sections prior to immunocytochemical processing. Storage of tissue in the solution at −20°C or 4°C for up to 3 months produced no adverse effects upon tissue morphology, nor was LHRH immunoreactivity diminished or accompanied by elevated non-specific staining. Furthermore, ultrastructural analysis of cryoprotected tissue revealed excellent preservation of cellular morphology. It is anticipated that this method can find use when it is necessary or desirable for the investigator to retain tissue for later immunocytochemical or electron microscopic processing.

References (5)

G.E. Hoffman et al.
Relationship of catecholamines and LHRH: light microscopic study
Brain Res Bull
(1982)
J.S. deOlmos et al.
The afferent connections of the main and accessory olfactory bulb formations in the rat. An experimental HRP study
J Comp Neurol
(1978)

There are more references available in the full text version of this article.

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Citation Excerpt :
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Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology

Abstract

Brain Res Bull

The afferent connections of the main and accessory olfactory bulb formations in the rat. An experimental HRP study

J Comp Neurol