Neuronal cultures on polyornithine coated glass coverslips - (Sep/09/2008 )
The title says it all - in order to use the 63x magnification of our microscope and to see the antibody-stainings I will do with my cortical neurons I need them plated on glass coverslips.
I already have a protocol for coating plastic dishes:
-place [x]µl polyornithine on dish
-incubate overnight at RT
-wash 3x with sterile water
This works quite well with the plastic dishes, so I wanted to take this for my coverslips. So after flaming the coverslips I place the PO on top of them, wait overnight, wash them and after weekend use them.
But it doesn't work the way I want it to be. Much cell-debris and dead cells float all around - the coating seemingly is not working correctly.
Does anyone do similar attempts, more successful than me, perhaps ?
My old lab uses poly-ornitine O/N and after the washes, add laminin and leave it O/N.
Thanks for your fast answer
But another question is following up We do not coat dishes or coverslips with laminin by default; although I would want to try this out right now. We only have some remnants of fibronectin. After a little research this could also be used to add more adhesiveness to my coverslips. But - now comes the point - the protocol attached to that from the nice person I got it from, is a little bit ... hm.
1. add PO, incubate O/N
2. wash with PBS
3. cover coverslips with PBS, incubate O/N
4. wash with PBS
5. add fibronectin
6. wait another night
7. wash with PBS
In contrast to that I found a protocol on Invitrogen-online which says to immediately add fibronectin after step 2.
I now wonder what the big difference is...? I mean, if coating takes two or three days, is a difference to me, personally but else...? Can I even use fibronectin for my cortical neurons, or is it just worthless for that?
We use Poly-D lysine.. put it on sterile (UV'd for 30 mins) coverslips, take it off after 20 mins and then 3 ten minutes washes with water.... leave to dry for 1 hour and its ready...works fine for neurons and astrocytes, etc.
Hello to all of you
I'd ask if someone could give me a complete protocol for culture DRG neurons from neonatal mice, I mean after they're isolated from vertebral column. We've just proved different procedures but anyone gave us good results.
Thanks so much