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

glial cells Immunofluorescence cover slip fixing

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#1 Sung Y

Sung Y


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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


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 : ) 


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

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