The Use of Microwaves for in vivo DNA Staining of Living Biological Samples
Background
This method provides a method to detect nuclear position and cell cycle evaluation on most cell types. Cells are not fixed so nuclei can be observed for under a variety of fluorescence conditions.
Reagents
NUCLEI STAINING BUFFER (NSB) (Based on Saxena et al. (1985).)
10mM MES.KOH (pH5.5)
0.2M sucrose
2.5mM EDTA
2.5mM DTT
0.1mM spermine
10mM NaCl
10mM KCl
20% glycerol
* For NSB + TRITON, make as above with the addition of 0.15% Triton X-100
Syto Staining Solution
Working dilution of 2 µM/mL-100µM/mL Syto green fluorescent nucleic acid stains in NSB or PB (Ca++ & Mg++ free) and (optional) 1mG/mL RNase A.
RNase A (optional):
Dissolve 1mG of DNase free RNase A in 1mL of DiH2O. (Note: Too much RNase will not effect the cells, however,
Lower concentration of RNase will effect the quality of analysis by preventing good nucleic acid staining.
Procedure
Make working dilution of Syto stain in NSB. Syto 16 (488 nm excitation, 518 nm emission) has been effective in Allium root tip nuclear staining at 50 µM/mL. Syto 62 at 50-75 µM/mL (652 nm excitation, 676 nm emission) is also effective in plant tissue staining using 647 nm line on Krypton\Argon laser systems for observation.
Prepare sample for observation using standard dissection techniques common to your system.
Place specimen into 16 cavity Teflon embedding mold (Ted Pella, Redding, CA). Any Teflon container (i.e. Teflon spot plate) should work fine.
Add NSB/Syto stain to cover specimen.
Teflon sample holder containing specimen is placed on the floor of a microwave vacuum chamber (cat. # 3435. Ted Pella, Redding, CA). The microwave temperature probe was placed through the probe hole of the chamber so that it rested in a sample holder well that contained only NSB (no tissue). The vacuum chamber was placed in the front of a model 3450 Microwave (Ted Pella, Redding, CA). A 15 mm Hg vacuum was pulled on the chamber with an oil-less vacuum pump (cat # 2Z866 Ted Pella, Redding, CA).
Samples were then exposed to microwave treatment for 5-15 min at a microwave power setting of approximately 250 watts (# 1 power level). The restriction temperature was set at 25°C for the entire process.
A gas permeable gasket was made using Carolina Observation Gel (cat # 63148-62-9, Carolina Supply Co., Burlington, NC). The specimen is then placed into the well and NSB added to fill cavity. A 22 x 22 mm #1 coverslip is then place over the sample and pressed gently to seal. We have had samples of various types remain viable for over one week under these conditions.
The coverslips can be viewed from either side. Specimens were observed using a BioRad 1024 Confocal attached to a Nikon Diaphot 300 inverted microscope. 4D images (3D over time) were recorded every 20 seconds over a 6-hour period. Images were stored to disk for later observation/analysis.
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