2.5. Gel microstructure
Images of the gel microstructure were taken using an inverted
confocal scanning laser microscope (Leica TCS SP2, model Leica
DM IRE2, Leica Microsystems CMS GmbH, Mannheim, Germany)
with an Ar/Kr visible light laser and 63 (oil) objective. Resolution
of the acquired digital images was 1024 1024 pixels. A fluorescence
dye (rhodamine B) (Fisher Sci., Fairlawn, NJ) (excitation
and emission wavelengths 543 and 625 nm, respectively) was used
for staining the protein. Twenty microlitre of rhodamine B (0.2% w/
v in milli-Q water) were added to 5 mL of sample for staining. Two
drops of sample were placed into grooves of a concave microscope
slide and a cover slip was placed over the top and sealed. Slides
were incubated at 40 C until pH 5.1 was reached, and then cooled
in a refrigerator for at least 30 min at 4 C before viewing under the
microscope.
2.6. Statistical analysis
Measurements were carried out in triplicate and treatments
were tested for equal variances then analysed using an unpaired
student’s t-test assuming equal variances, using the using the data
analysis function in Microsoft Office Excel 2007.