Microwave-Assisted Processing
continuous microwave radiation
in combination with no
microwave-induced sample
heating. Microwave devices for
the present research were from
the PELCO BioWave
®
line of
microwave technology (Ted Pella,
Inc. Redding, CA).
Results
For the four techniques that
clearly benefit from the presence
of microwave radiation (EM
fixation and tissue processing,
immunolabeling, formaldehyde
fixation and decalcification), a
reduction in turnaround times
Figure 2: (A) Kidney glomerulus fixation results for a 24-hour conventional fixation with 2.5% glutaraldehyde in 0.1M
sodium cacodylate buffer compared to (B) 24-hour fix in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.06M
has been a significant component.
phosphate buffer. Both primary fixations were followed by microwave-assisted fixation in 2% aqueous osmium These time savings can be
tetroxide and processed to polymerized resin blocks by the microwave methods of Giberson et al. (2003). When both
summarized as follows:
primary fixations are microwave-assisted, the results mimic B (no shrinkage seen in the glomerulus or between tubules
as seen in A). The asterisks in A and B denote Bowman’s Capsule. Scale bar = 2 µm.
• EM processing (fixation
through resin infiltration): reduced
on the bench is freely transferable to a microwave-processing
to < 40 minutes
environment. This is especially true for fixatives containing
• EM resin polymerization (epoxy resins and LR White):
formaldehyde. Galvez et al. [2] elucidated the role that wattage
reduced to < 75 minutes
and sample temperature control play in microwave-assisted
• Single labeling protocols (fluorescence or DAB): reduced
fixation of fresh tissue in formaldehyde. These same factors
to < 60 minutes
were also the key components to reproducible microwave- • Formaldehyde fixation for histology (tissues <5mm
assisted immunolabeling [4]. The significance of true wattage on thick) reduced to 60 minutes
embryo viability after microwave exposure was demonstrated • Decalcification with EDTA (based on conventional
experimentally by Sanders and Gartner [8]. Early microwave- methods): >10X rate increase.
assisted processing techniques for electron microscopy did not Consistent fixation results are concurrent with the
have the advantage of true wattage or control of the microwave noted time savings. Figure 1 demonstrates the effect of 20
environment as did the later techniques [3, 6]. Tingling et al. [6] minutes of microwave exposure on the rate of ultrastructural
used a microwave-assisted technique for EDTA decalcification preservation with 10 percent neutral buffered formalin.
that controlled sample temperature external to the microwave Microwave involvement resulted in a significant improvement
device being used. This was the first demonstration of in ultrastructural detail when compared to 3 hours on the
Figure 3: HeLa cells transfected with CellularLights, tubulin GFP (Invitrogen, Carlsbad, CA) fixed in 3% paraformaldehyde and then labeled with an anti-GFP antibody
followed by Alexa 488 secondary detection. (A) Cells fixed conventionally for 30 min. at 37 C. The continuity of label down the microtubules is not uniform (arrows). (B)
Transfected Hela cells fixed in the presence of 150 W of microwave radiation for 1 min. at 37 C. The continuity of the label is superior as compared to A above. Bar = 2 µm.
30
www.microscopy-today.com • 2009 September
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