Preparation of coverslips: At Lonza we used coverslips from Menzel (part of Thermo Scientific; http://www.menzel.de/Deckglaeser.675.0.html?id=675&L=1). We think that the coverslips have such a good quality that they don’t have to be cleaned prior to use.
If you want to use other coverslips or prefer cleaned coverslips, we would recommend the protocol from Zeitelhofer et al., 2007, Nature Protocols:
- Place coverslips in porcelain staining racks and submerge in 65% nitric acid for 18–36 h. Wash coverslips in sterile, distilled and deionized water three times for 5 min followed by three times for 20 min.
- Place the porcelain racks with the coverslips in a glass container and dry in an oven at 70 °C (typically it takes 1 h). PAUSE POINT: These clean coverslips can be stored in a dry, sterile environment for up to 2 weeks.
- Cover the glass container holding the porcelain racks with the coverslips with aluminum foil and sterilize in an oven with dry heat at 220 °C for 7 h.
CRITICAL STEP: Do not autoclave the coverslips, as this will render them unsuitable for neuronal culturing.
NOTE: The optimal coating substance depends on neuron type; the coating described below was successfully used at Lonza for rat/mouse cortical and rat hippocampal neurons.
- Prepare a stock solution of 1 mg/ml poly- L- Ornithine (e.g. Sigma: Cat# P3655-50mg) in distilled water. Store in aliquots at -20°C or as recommended by the supplier.
- Freshly dilute the stock solution 1:10 in distilled water to obtain a 100 µg/ml poly- L-Ornithine working solution.
- Use 500 µl/well (containing a glass coverslip). - Put back the lid onto the plate and incubate over night at room temperature (~22°C). - Wash twice with 1 ml of distilled water and let dry (open lid) under a sterile hood for approx. 2 hours.
- Seed neurons If it generally works, you may reduce coating concentration to 10µg/ml. If it does not work well, you could try 1 mg/ml or switch to poly-D-lysine or additional laminin-coating. If you need further support in terms of coating it would be great if you could send us some pictures for troubleshooting.