Jacobs:Protocol Coating Slides with Fibronectin
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Coating Slides with Fibronectin
- Fibronectin (Sigma #F1141-5mg)
- Petri dishes
- Microscoope slides 75 x 38 x 1 mm (Fisherbrand #12-550B)
Day 1 (~1 hr with 1 hr waiting)
- Slide preparation
- Soak slides in 100% EtOH for at least five minutes (usually 20 for flow is a good number).
- Open 20 petri dishes in the hood.
- Bring the slides and EtOH into the hood.
- Sterilize the flat-ended forceps in the same 100% ethanol.
- Lean slides to dry on edge of the petri dish (face up)
- Flip the slides after laying them all down so that the other side can dry and any ethanol that pools on the lid can dry.
- Once all the ethanol has evaporated (there should be no wet shiny places on the slides), place the slide flat on the bottom of the petri dish.
- Place covers on the dishes.
- Prepare 10 ul fibronectin per 1 mL PBS solution (1:100). You will need 1 mL of solution per slide.
- Volume of Fibronectin solution = [# of slides] x 1 ml/slide
- Store for up to 6 months at 4º C
- For 20 slides, that means 200 ul fibronectin into 19.8 ml PBS.
- Mix well.
- Carefully pipette 1 ml of fibronectin solution on top of each glass slide. This is a delicate process, be careful and take it slow
- Do not allow the solution to fall over the edge of the slide. If this happens, you need to collect the liquid, sterilize the slide, dry and try again. Any liquid that falls over the side will suck the rest of the liquid out and under the slide, which we do not want.
- Make sure the solution covers the entire slide. You may use a sterile pipette tip to smooth the liquid onto any bare spots. Surface tension will allow the liquid to stay on the slide, but again, don’t push it over the sides.
- Cover and let the slide sit undisturbed at RT in the cell culture hood for 1 hr.
- Plate cells
- FAK is expressed at high levels in FAK +/+ fibroblasts. The FAK-related kinase, Pyk2, is expressed at low levels in FAK +/+ fibroblasts and at high levels in FAK -/- fibroblasts. Upon replating on fibronectin, both FAK and Pyk2 will form transient signaling complexes with the c-Src protein-tyrosine kinase. Replating of cells on fibronectin can also lead to the activation of c-Src through dephosphorylation of Tyr-527 located in the C-terminal domain of the regulatory region. This protocol describes the procedure for coating microscope slides with fibronectin.