After amplification, the well contents were removed, and the wells were washed six times with 200 [micro]L TBST
with a 5-min soak step after the third wash.
To detect digoxigenin-labeled amplicons hybridized with biotinylated probes, 200 [micro]L of anti-digoxigenin-horseradish peroxidase (HRP)-conjugated antibody (10 U/L in TBST
; Roche Molecular) was added and incubated at 25[degrees]C for 30 min; the black plates were then washed five times with TBST
(4) Nonstandard abbreviations: CA VI, carbonic anhydrase isoenzyme; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; and TBST
, Tris-buffered saline with Tween-20.
3 mg chicken embryo powder was added to 1 ml TBST
After blocking for 2 h by 5% skim milk in TBST
(10 mM Tris-HC1pH 7.5, 150 mM NaCl, 0.1% Tween-20), the membrane was incubated for 2 h with anti-ABCA1 or anti-Flotillin2 antibody.
The membranes were blocked with 5% non-fat milk in TBST
(Tris-buffered saline with 0.1% Tween 20) for 1 h, washed and then incubated with the following primary antibodies: anti-PARP, anti-caspase-3, anti-Cyclin Di, and anti-Bcl-2 (diluted 1:1000 in 5% skim milk in TBST
; Santa Cruz Biotechnology), anti-VEGF (diluted 1:500 in 5% skim milk in TBST
; Santa Cruz Biotechnology).
The filters were blocked in TBST
containing 5% nonfat milk powder overnight and then incubated for 2 h with a 1:50-diluted anti-human PA28gamma (Mid) rabbit polyclonal antibody (Zymed Laboratories, USA) in TBST
containing 5% nonfat milk powder.
The membranes were blocked in 5% nonfat dry milk in Tris-buffer saline containing 0.05% Tween 20 (TBST
) for 2 h at room temperature.
The membranes were washed in Tris-buffered saline with Tween 20 and incubated in 5% skim milk (Sigma, USA) at room temperature for 2 h on a rotary shaker, followed by TBST
washing three times.
The membranes were blocked for 30 min using TBST
(20 mM Tris, 137 mM NaCl, and 0.1% Tween-20 detergent (pH 7.6)) with 5% skimmed-milk powder, and then, probed overnight at 4 [degrees]C with rabbit anti-CctA antiserum (FREY et al., 2012) diluted 1:1000 in TBST
, followed by incubation for 2 h at room temperature with phosphatase-labelled goat anti-rabbit (KPL 4751-1516) diluted 1:5000 in TBST
The membranes were blocked with 5% non-fat milk for 1 h at room temperature, washed three times with Tris-buffered saline with Tween-20 (TBST
) and incubated with p-STAT3 (1:1000), STAT3 (1:1000), COX-2 (1:1000), MMP-2 (1:1000), MMP-9 (1:1000), N-Cadherin (1:500), E-Cadherin (1:500) and GAPDH (1:1000) antibodies overnight at 4AdegC.
The membranes were blocked in tris-HCl buffer (TBS) containing 0.1% Tween 20 (TBST
) containing 5% nonfat milk at room temperature for 45 min.