Paraffin slices were dewaxed and rehydrated, then stained with hematoxylin (Beyotime Biotechnology, China) for 3 min, washed for 5 min, destained
with hydrochloric acid alcohol, washed for 5 min, and fast stained with eosin (Beyotime Biotechnology, China).
5% agarose gel for 30 minutes at 100 volts, stained with ethidium bromide, destained
and visualised by UV transilluminator.
The band of interest was excised, destained
by 5 washes in 10% acetic acid, washed 3 times in deionized water and air-dried.
125% (m/v), 40% (v/v) 2-propanol, 10% (v/v) acetic acid] and destained
[40% (v/v) 2-propanol, 10% (v/v) acetic acid] until optimal band patterns were observed.
Protein spots of interest were cut from the preparative gels, destained
for 20 min in 30 mM potassium ferricyanide/100 mM sodium thiosulfate (1:1 v/v) and washed with Milli-Q water until the gels were destained
1% (w/v) Coomassie brilliant blue R-250 (Sigma), 30% (v/v) methanol and 10% (v/v) glacial acetic acid solution and destained
in a solution containing 30% (v/v) methanol and 10% glacial acetic acid in distilled water.
Slides were incubated for 1 h at room temperature in phosphoprotein stain (Invitrogen #MP33300; Thermo Fisher Scientific) while protected from light, destained
for 3 x 30 min with a destaining solution specific to the stain (Invitrogen, #P33310; Thermo Fisher Scientific), and washed again for 2 x 10 min in ultrapure water.
Protein spots were excised from the gel and destained
in a solution of 50 mM sodium thiosulfate and 15 mM potassium ferricyanide till the color disappears.
After counting, plates were destained
with methanol, and absorbance values were measured at 584 nm using a FLUOstar OPTIMA microplate reader (BMG Labtech Inc.
125% (w/v) Coomassie brilliant blue for 20-30 min and destained
in destain buffer (10% acetic acid and 5% ethanol in distilled water) for 1-2 days.
The slides were then washed and destained
for 5 min in distilled water and examined immediately under fluorescence microscope.
Slides were then destained
with a series of alcohol baths (100%, 95%, and 70%, 3 minutes each) and then submerged in ice-cold Carnoy solution for 10 minutes, followed by phosphate-buffered saline for 5 minutes and air dried.