Microwave-Assisted Processing
Figure 4: Fluorescence microscopy of living GFP/nRap1 expressing zebrafish embryos before (A) and after (B) fixation with 3% paraformaldehyde and 0.5%
glutaraldehyde in PBS. Conventional fixation times were evaluated from a few minutes up to an hour with similar results. The GFP fluorescence was maintained when
the fixation was microwave-assisted (2 minutes at 150W, 2 minutes at no power, followed by 2 minutes at 150 W). Images A, B, and C are all at the same magnification.
Scale bar in C = 0.15 mm. The GFP expression in embryos following microwave fixation in C is shown at a higher magnification in D (bar = 50 µm).
bench at the same temperature [4]. The evaluation of the importance of a microwave-assisted fixation is demonstrated
results from a 24-hour bench fixation with two EM fixatives, in Figures 4A-D. A conventional fixation results in the loss of
paraformaldehyde/glutaraldehyde and glutaraldehyde alone, the GFP fluorescence (Figure 4B). When the same fixation is
indicate the difficulty in standardizing fixation with time microwave-assisted, GFP fluorescence is maintained (Figures
(Figure 2). The study indicated that a 24-hour time period 4C-D).
produced the best results for the mixed aldehyde, whereas 2
hours was best for glutaraldehyde alone (results not shown).
Discussion
The shrinkage artifacts seen in Figure 2A were eliminated when
The real benefits of microwave-assisted methods go
the fixation was done in the microwave, and the quality of
beyond time savings. Those benefits, we demonstrate, reside
fixation for the mixed aldehyde was maintained as well (Figure
in a microwave-assisted fixation step (Figures 1-4). Microwave
2B) [4,7].
methods provide the tool to standardize the fixation process
Figures 3A-B present the results of a conventional fixation
to a matter of minutes versus hours, and the benefit of that
and labeling contrasted with a microwave-assisted fixation and
approach can be seen in Figure 2. It is further demonstrated
labeling protocol. HeLa cells transfected with Cellular Lights
by high consistency in immunohistochemistry (Figure 3).
Tubulin-GFP (Invitrogen, Carlsbad, CA) were conventionally
Figure 4 provides further evidence of the uniqueness of
fixed for 30 minutes (Figure 3A) or 1 minute at 150 W true
microwave methods. GFP fluorescence is maintained only after
wattage (Figure 3B). The microwave-assisted processes (fixation
a microwave-assisted fixation (Figure 4).
and labeling) resulted in improved immunohistochemistry as
Improved microwave technology has provided the ability
evidenced by the continuity of the microtubule structure. The to define and control the important experimental parameters
2009 September •
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