Neurosphere attachment - Neurosphere attachment (Sep/07/2005 )

Dear reader,

I am quite frustrated as my supervisor is not allowing me to proceed unless I overcome the problem of attachment/immunostaining of cell aggregates.

I read many papers whereby Poly-L-ornithine or Poly-L-Lysine is used to coated glass coverslips prior to laminin coating. (I tried this but did not work for me.) Alternatively, gelatin is used for coating glass coverslips. The gelatin coating works well whereby the cells aggregates have a high attchment of 80% but of these, only 6% remains after immuno processing.

What I am eager to know is; how to coat glass coverslips such that cells aggreates/neurosphere/embryonic bodies remain firmly attached?

How to immunostain cells aggreates/neurosphere/embryonic bodies such that no loss of sample occurs?

Anybody has a clue about what I am talking?

Many thanks

Frustated researcher ...

Try this.

Make a 10ug/mL poly-l-lysine solution in water, coat your coverslips for 1 hr. wash twice for 10 min each with water, than 10 minutes with media (straight from the bottle, doesn't need additives). plate your spheres. they should attach nicely.

however, as far as i know there is NO way to get 100% recovery, you will ALWAYS lose spheres during staining. oftentimes >30%. deal with this by plating more spheres, or by letting them differentiate for a few extra days to attach better.

you need to wash the PLL before adding the laminine

we coat with p-orn+ lam and have tried to resolve this by doing very gentle partial rinses for all the staining steps. Of course, partial rinsings results in higher background.

We use Poly L coverslips for Drosophila CNS immunostaining and suggest the following tips:

Don't reuse the Poly-L solution more than 3 or 4 times, as it loses stickiness, and likewise, don't use the slips after a couple of weeks, again the stickiness issue. This may help?

you might want to try acid washing your coverslips prior to coating with poly-lysine etc.
In a fume hood put the coverslips in Nitric acid overnight (being careful with the concentrated acid !). Wash with 4 changes of water and sterilise coverslips before coating.

hope this helps