Tissue vitrification using an 8.4 M cryoprotectant cocktail solution (VS55) was achieved in 1 ml samples by imposing a low (2.6 ± 0.1 °C/min) cooling rate, between −40 and −100 °C, and a low rewarming rate (62 ± 4 °C/min) between −100 and −40 °C.
The culture was initiated by inoculation of 2.8% of culture volume into the prepared culture medium containing 50 μg/mL of kanamycin (Sigma, St . Louis MO, USA), 7.6 g/L of trace metal cocktail solution, 15.8 g/L of glycerol (Sigma, St . Louis MO, USA) and 1% (v/v) P2000 antifoam (Alfa Aesar, Reston, VA, USA) solution.
Neutralized rat tail type I collagen (2 mg/mL, BD, Biosciences, Palo Alto, CA, USA) was suspended in medium cocktail [DMEM, NaHCO3 (44 mmol/L), L-glutamine (4 mmol/L), Folic Acid (9 mmol/L), and neutralized with 1 N NaOH to pH 7.2]. 1 × 105 cells were suspended in 1 mL collagen and medium cocktail solution and plated on filter inserts.
Cells were collected and lysed in a lysis buffer (Cell Signaling) supplemented with 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail solution (Sigma).
To obtain whole protein lysates, cells were incubated with radio immune precipitation assay buffer (containing 100 µl/ml of a protease inhibitor cocktail solution [Roche, Penzberg, Germany]) and incubated on ice for 30 minutes.
Using protocol described [52], rats were perfused with 4% paraformaldehyde and brains were cut into frontal sections (40-µm thick), which were then incubated in a cocktail solution containing mouse anti-GluR2 antibody (1∶100; Chemicon) and sheep anti-TH antibody (1∶100; Chemicon) or rabbit anti-GAD65/67 anti-GAD65/6700; Chemicon) for 3 dantibody
Cocktail, cocktail, cocktail, cocktail......
Few cytotoxicity studies have investigated vitrification-relevant concentrations of CPAs, and the benefits and disadvantages of cocktail solutions and of incorporating non-permeating solutes have not been fully evaluated.
One microliter of the cocktail solutions per spot was transferred to a dry filter paper.
The protecting groups were cleaved by treatment of the dry resin with the resin cleavage cocktail stock solution (2.50 mL) for 2 h at room temperature with vigorous stirring.
The resin was filtered and rinsed with the remaining resin cleavage cocktail stock solution (1.50 mL) and TFA (1.50 mL), collecting the filtrate and rinses in a 50 mL BD Falcon tube.
