Electrophoresis was performed at a constant voltage of 120 V for 90 min. Gels were washed in a re-naturing buffer and incubated in an incubation buffer at 37°C for 24 h, stained with Coomassie brilliant blue R-250 (Sigma), and then de-stained with gel-clear de-stain solution.
For BrdU detection, deparaffinized sections were microwaved in citrate buffer and incubated in HCl prior to incubation with primary antibodies.
At the end of the incubation, the plates were washed three times with washing buffer and incubated in diluted secondary antibody, biotin-labeled anti-human TNF-α polyclonal antibody (200 ng/mL), for 1 h at 37°C.
Subsequently, embryos were washed three times in washing buffer and incubated in 1 200 goat anti-rabbit Alexa Fluor 488 (Molecular Probes) solution in blocking buffer overnight at 4 °C.
The cells were then washed with wash buffer and incubated in the dark for 30 min, followed by nuclear staining with 5 ng ml−1 DAPI in wash buffer and another 30 min in the dark, and finally a wash with 2×SSC.
Samples were permeabilized in 0.1% Triton-X in PBS for 15 minutes at room temperature, washed in blocking buffer, and incubated in 1 100 tubulin-FITC (Cell Signaling, Beverly, MA) and 1 50 rhodamine phalloidin (Life Technologies) overnight at 4 °C.
The enzymes were added separately to 10 mL hydrolysate in 250 mL Erlenmeyer flask along with 25 mL of 0.1 M citrate buffer and incubated in reciprocal shaker at 50 °C at 120 rpm for 24 h.
The following day the cells were washed in 0.1 M Na cacodylate buffer and incubated in 1% osmium tetroxide (Mecalab) for 1 h at 4°C.
The sections were then washed with buffer and incubated in 0.05% DAB (3,3'-diaminobenzidine; Sigma) and 0.03% H2O2 in phosphate buffer at room temperature.
