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Pretreatment methods to improve nerve immunostaining in corneas from long-term fixed embryonic quail eyesPretreatment methods were used to improve neurofilament immunostaining in corneas from embryonic day 16 Japanese quail corneas that had been stored in fixative solution for several months. A sequential combination of the following three pretreatments: brief microwave heating in saline, followed by extraction with sodium dodecyl sulfate (SDS) at 37 degrees C, followed by digestion with hyaluronidase at 37 degrees C, produced significantly increased antibody staining of corneal neurofilament proteins, compared with embryonic corneas subjected to no prior pretreatments or to single or two-step protocols. After applying the sequence of all three pretreatments, darkest nerve staining and increased numbers of fine branches were observed, together with lower background staining. Thus, the result of applying the three-step pretreatment sequence is better than that of applying any of its component single pretreatments or even combinations of any two of them. These findings therefore suggest that each of these three pretreatments causes a unique effect, beneficial to immunostaining of neurofilament proteins, and that their individual effects are independent and additive. In addition to embryonic corneas, the three-step procedure also may be useful for immunostaining of nerves in other very delicate, highly-hydrated tissues containing an abundance of extracellular matrix.
Document ID
Document Type
Reprint (Version printed in journal)
Barrett, J. E.
(Kansas State University Manhattan 66506-4901, United States)
Wells, D. C.
Conrad, G. W.
Date Acquired
August 22, 2013
Publication Date
October 15, 1999
Publication Information
Publication: Journal of neuroscience methods
Volume: 92
Issue: 2-Jan
ISSN: 0165-0270
Subject Category
Life Sciences (General)
Funding Number(s)
Distribution Limits
NASA Discipline Developmental Biology
Non-NASA Center
NASA Program Fundamental Space Biology

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