poly-D-lysine slides

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poly-D-lysine slides

Postby rlamina » Jun 23 2008 3:38 pm

Hi all,

I work with several epithelial lines that have to be transfected and treated for immuno-fluoreescence, for this reason I have to seed them directly on glass coverslip into a 24 well plate. Generally this cells tend to detach during the immunofluoreescence treatment, I've heard that coverslips can be covered with poly-D-lysine in order to make them more "sticky ", does anyone of you know this protocol? Do you know which kind of poly-d-lysine i have to use?

Thanks in advance
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Postby Astarte » Jun 24 2008 8:43 am

protocol for poly-d-lysine coating :'s%20protocols/Coating%20Coverslips.htm

You can also try coating with collagen, it's cheaper than poly-d-lysine and also works for most cells.
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Re: poly-D-lysine slides

Postby steveneam » Jul 03 2010 12:28 pm

Sorry, but would you have the protocol for collagen coating? I've also heard about gelatin. But both are extremely hard to dissolve in PBS I think.
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Re: poly-D-lysine slides

Postby CrowSan » Jul 07 2010 10:48 am

There are a few protocols for poly-Lys treatment (check http://www.ihcworld.com/smf/index.php?topic=2332.0).
Collagen treatment is fairly simple I have pasted the protocol from BD Biosciences Collagen below.

Gelling Procedure- Rat Tail Collagen HC will gel when its pH is brought to alkalinity using the procedure below.
Please use this as a guideline for determining the optimum concentration for your application.
1) Prepare ammonia vapor chamber by taping a sterile 2 inch gauze sponge to the inside lid of a 150mm petri
dish. Saturate the gauze with ammonium hydroxide. Place lid on 150 mm dish and set aside.
2) Place an even coating of collagen on surface to be coated. Thickness may be varied as desired. 50 to 100
ul of collagen is sufficient to coat a 22mm coverslip. For dishes of 100mm diameter, add approximately 6.0
ml per dish; for 60 mm dishes add approximately 2.3 ml, and for 35 mm dishes add approximately 1.0 ml.
3) Transfer coated coverslips or dishes with lids off to ammonia vapor chamber and expose for three minutes.
4) Soak coated coverslip or dishes in sterile dH2O for 30 minutes (5 ml for 35 mm dishes, 10 ml for 60 mm
dishes, etc.). Aspirate and replace with 0.5 to 1 ml of sterile dH2O and let sit overnight lidded in a laminar
flow hood.
5) Aspirate the dH2O and replace with serum supplemented balanced salt solution and store at 2-8°C.

Hope this helps

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