Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase.

Abstract

Adequate fixative in immunohistochemistry requires not only a rapid and total immobilization of the antigen, but also a sufficient preservation of its immunoreactivity and maintenance of its accessibility to the immunochemical reagents for localization. Thus, the optimal fixation condition for a specific antigen necessitates a compromise between these opposing variables and can be determined by the preparation of a series of tissues with a progressively increasing degree of fixation. Unless the results of localization using such a series is available, one must be satisfied with adequate but less than optimal results. In the present study, this principle is demonstrated using the localization of tyrosine hydroxylase in the dopaminergic system with formaldehyde as the fixative. The rate and degree of fixation with formaldehyde was shown to be highly pH dependent. By perfusing the tissue with formaldehyde at pH 6.5 (where the rate of fixation is extremely slow) it is possible to rapidly distribute the fixative homogeneously into the tissue. By suddenly changing to a formaldehyde perfusate of higher pH, the cross-linking reaction is rapidly increased. This two-step fixation procedure provides a means of obtaining a rapid and uniform immobilization of the antigen, so that its translocation can be avoided. The final degree of fixation is controlled by the duration and pH of the second fixative solution. The results obtained by increasing the pH of the second solution demonstrated that complete fixation of tyrosine hydroxylase in the dopaminergic system with formaldehyde maybe obtained using a very basic formaldehyde solution (pH 11) while still retaining immunoreactivity of the enzyme. The localization that was achieved at lower pH appeared adequate until it was compared to the results obtained by perfusion at pH 11 in the second step.

Identification of cDNA clones coding for rat tyrosine hydroxylase antigen.

“…(iii) Dopamine-3-hydroxylase and phenylethanolamine-N-methyltransferase activities from rat adrenal glands were not inhibited by this antiserum. (iv) Immunohistochemical studies indicated that it specifically stained catecholaminergic neurons (34,35). This antibody crossreacts with rat TyrOHase and, although it has been raised against a 40,000-dalton moiety, the 62;000-dalton protein characteristic of rat TyrOHase was immunoprecipitated from a PC 12 cell lysate.…”

Evidence that the bifunctional redox factor / AP endonuclease Ref-1 is an anti-apoptotic protein associated with differentiation in the developing retina

“…42 Both protocols produced similar results, but the quality of both fixation and immunohistochemistry was the best with the sequential procedure at variable pH. The eyecups containing the retina in situ were oriented to allow sagital sectioning, and cut frozen at 10 mm thickness, after removal of the cornea and lens.…”

An Ultrastructural Analysis of the Distribution of Angiotensin II in the Rat Brain

“…Rats were deeply anaesthetized with sodium pentobarbital (40 mg/kg) then perfused transcardially with a brief saline wash, followed by two different aldehyde solutions in a similar manner to that described by Berod (38). The first of these contained 4% paraformaldehyde in a 0.1 M sodium acetate buffer (pH 6.5) and the other, 4% paraformaldehyde and 0.05% gluteraldehyde (PH 9.5) in a 0.1 M sodium-borate buffer.…”