Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Immunofluorescence - Cover Slip and Fixing problems with Hs683 (glial) Cells

glial cells Immunofluorescence cover slip fixing

  • Please log in to reply
No replies to this topic

#1 Sung Y

Sung Y

    member

  • Members
  • Pip
  • 1 posts
1
Neutral

Posted 03 February 2014 - 01:33 PM

Hi all, 

Glial cells are not sticking as well as I hoped on the coverslip.

 

General Protocol:

1) Used a 22-mm glass coverslip in a 6 well plate. 

2) Used 2 ml poly-L-lysine in sterile water to cover the entire coverslip

3) Incubated it for 15 minutes

4) Washed with PBS 

5) Placed it under UV to sterilize the coverslip

Result:

Most cells only grew on the outer edges of the coverslip, but very few grew in the center. 

Maybe there was grease? But I washed it with ethanol before I did anything.

 

Also, after I fixed the cells and observed them through the microscope, I found that all of my cells were "ripped apart". I barely saw any intact glial cells. 

General Protocol: 

1) Fixed 4% paraformaldehyde for 20 min at 4°C

2) Washed with PBS

3) Permeabilize with 0.2 % Triton X-100 in PBS for 5 mins at RT

4) Washed with PBS

5) added goat serum to block non-specific staining, added primary antibody, washed with PBS, added secondary antibody (AlexaFluor, binds to protein in nucleus), washed with PBS, stained with DAPI, added Mountant... and so on

 

I couldn't see any glial cells that had their overall structure. 

Do I need to use a different reagent for fixation? Maybe glial cells are really sensitive...so should I be extra careful next time?

Please let me know what you guys think : ) 

-Sung 


Edited by Sung Y, 03 February 2014 - 01:36 PM.






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.