Two-color fluorescence labeling in acrolein-fixed brain tissue

Abstract

Acrolein is a potent fixative that provides both excellent preservation of ultrastructural morphology and retention of antigenicity, thus it is frequently used for immunocytochemical detection of antigens at the electron microscopic level. However, acrolein is not commonly used for fluorescence microscopy because of concerns about possible autofluorescence and destruction of the luminosity of fluorescent dyes. Here we describe a simple protocol that allows fine visualization of two fluorescent markers in 40-mum sections from acrolein-perfused rat brain. Autofluorescence was removed by pretreatment with 1% sodium borohydride for 30 min, and subsequent incubation in a 50% ethanol solution containing 0.3% hydrogen peroxide enhanced fluorescence labeling. Thus, fluorescence labeling can be used for high-quality detection of markers in tissue perfused with acrolein. Furthermore, adjacent acrolein-fixed sections from a single experiment can be processed to produce high-quality results for electron microscopy or fluorescence labeling.

Figure 1

Effect of sodium borohydride pretreatment on unlabeled acrolein-fixed sections. Representative confocal microscopy stacks from pairs of adjacent sections from different animals, obtained with the same scanning settings, are shown. (A,C) show strong tissue fluorescence in non-treated sections. (B,D) NaBH₄-treated sections display no tissue fluorescence. Note that sporadic autofluorescent blood cells adhered to the blood vessel wall are still observed after the treatment (A,B, arrows). Putative endothelial cells also displayed autofluorescence before and after incubation in NaBH₄ (C,D, arrowheads). Bar = 20 μm.

Figure 2

Graphs show mean intensity values in NaBH₄-treated (gray) vs non-treated (black) unlabeled sections from five animals. Mean intensity values ranged from 0 (black) to 255 (maximum intensity). (A) Values obtained in the green channel. (B) Values obtained in the red channel. R number = rat subject number.

Figure 3

Effects of incubation in ethanol plus H₂O₂, ethanol only, or H₂O₂ only, on two-color fluorescence labeling. (A–F) Merged images from confocal microscopy of double-labeled sections from the lateral dorsal tegmental nucleus showing cholinergic neurons in red and BDA-labeled fibers in green. All images were obtained with the same acquisition settings. (A,B) sections from rat R175 without (A) and with (B) incubation in a 50% ethanol plus 0.3% H₂O₂, prior to the two-color fluorescence labeling. (C–F) Four serial sections from rat R194 showing the effect of different incubation solutions: (C) PBS; (D) water plus 0.3% H₂O₂; (E) 50% ethanol only; (F) 50% ethanol 0.3% plus H₂O₂. BDA, biotinylated dextran amine. Bar = 20 μm.

Figure 4

Effect of incubation in ethanol plus H₂O₂, ethanol only, or H₂O₂ only, on autofluorescent cells in NaBH₄-treated acrolein-fixed sections. (A–D) Serial sections showing the same longitudinally sectioned blood vessel, incubated in different solutions: (A) PBS; (B) water plus 0.3% H₂O₂; (C) 50% ethanol only; (D) 50% ethanol 0.3% plus H₂O₂. Blood cells were visible in the four different conditions but were generally associated with remnants of blood vessel wall (A–D, arrows). Autofluorescent endothelial cells were also observed in the four conditions but were scarcer than blood cells (e.g., arrowheads in A and D). (E,F) Images of a single section incubated in ethanol plus H₂O₂, obtained in the green (E) or red (F) channel, showing a putative endothelial autofluorescent cell (arrowheads) that displays a stronger luminosity in the green channel. Bar = 50 μm.