Cells were treated with the drugs at a series of concentrations and stained with sulforhodamine B, a dye that binds stoichiometrically to all cellular protein components, as a measure of cell proliferation (Vichai and Kirtikara, 2006).
The sections were dehydrated with a graded series of increasing ethanol concentrations and stained with hematoxylin-eosin.
Briefly, HaCaT cells were cultured overnight in black 96-well plates, incubated for 24h with each NCE at the indicated concentrations and stained with cytotoxicity cocktail.
Sections were de-waxed in xylene, rehydrated through a graded series of alcohol concentrations and stained with hematoxylin and eosin.
The samples were fixed with electron microscopy-grade paraformaldehyde (4% final concentration) and stained with SYBR Gold (1× final concentration; formally Molecular Probes, Inc., now Invitrogen, Solana Beach, CA) and filtered onto 0.02 µm Anodisc filters (Whatman, Inc, Florham Park, NJ), mounted on glass slides and directly counted by epifluorescence microscopy.
Since proteoglycans have many closely placed negative radicals, sections with high concentrations of proteoglycans, stained with toluidine blue, determine closely arranged and interacting molecules of this stain, replacing the blue with a violet coloring.
The sections were first treated with 0·5 M ethylenediamine tetraacetic acid (EDTA) for 3 min, dehydrated with graded concentrations of ethanol, stained with HistoGene Staining Solution [LCM Frozen Section Staining Kit (Arcturus®; Applied Biosytstems, cat. no. KIT0401NS, Foster City, CA, USA)] for 45 s.
Thereafter, cells were fixed with TCA, final concentration 10% and stained with SRB (0.4% wt/vol In 1% acetic acid).
For DIGE experiments, 50 µg of protein was adjusted to pH 8.8 by adding 50 mM Tris HCl final concentration, and either stained with 400 pmol of Cy3 or Cy5 (GE Healthcare).
Cells were treated for 24 h with different concentrations of bortezomib, then stained with annexin V-Cy5.
