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Author Topic: Cells detachment after fixation  (Read 8756 times)

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Offline SY

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Cells detachment after fixation
« on: September 05, 2009, 08:13:29 PM »
Hi,

I have problem with cell detachment after fixation (using 4% PFA/sucrose) on dissociated hippocampal cultures maintained in DMEM. I have previously been fixing the cells using the same method in cultures maintained in Neurobasal and have not encountered the same problem.

Could it be something to do with the osmolality of the fixative medium? Does anybody have the same problem before? Any advice is greatly appreciated.

SY

Cells detachment after fixation
« on: September 05, 2009, 08:13:29 PM »

Offline funk.106

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Re: Cells detachment after fixation
« Reply #1 on: September 08, 2009, 04:58:29 PM »
I used to do ICC on dissociated neuron cultures that had been grown on glass coverslips in a sandwich culture format. It's hard to say what may be going on because you don't give a lot of details of your protocol, and it has been a while for me since I've done it, but this is the protocol that I used to use. Also, I remember that I used to treat the coverslips with poly-d-lysine to help with attachment to the coverslips, so you could try this if you are not already doing so.

1)   With help of a forcep transfer the neuron plated coverslip (15mm in diameter) from culture plate to a PBS (pH 7.4) containing well of a 12 well plate.
2)   To fix the neurons aspirate off the PBS using vacuum and add 1ml of 4%PFA 1%Sucrose solution (prepared in 1X PBS) to the coverslip making sure the cells are fully covered with the fixative. Close the lid of the plate. PFA fumes are toxic.
3)   Fix for 13-15 minutes and wash (aspirate off using vacuum) the coverslip three times with PBS.
4)   Wait for 30 minutes at least to re hydrate the neurons properly and then check the fixed cells under the microscope to decide whether fixation has been done correctly or not. 
6)   Give 3 quick washes with PBS.
7)   Block the coverslip with 10% BSA (prepared in 1X PBS) for 45 min to 1hr at room temperature. 200ul is sufficient to cover the entire surface of one 15mm diameter coverslip.
8)   Rinse the coverslips once with PBS.
9)   Add 1 Antibody in 1% BSA and incubate overnight at 4C. After the overnight incubation, set the plate at room temperature and let incubate for about one more hour. 
10)   Give three 10 minute PBS washes
11)   Add 2 antibody and incubate for 1 to 2 hrs. 200l is sufficient to cover the entire surface of one 15mm diameter coverslip
12)   Give four 10 minute PBS washes.
13)   Label the slides on which the coverslips will be mounted
14)   Add 7 to 10ul of Vecta Shield on the frosted side of the slide. Take the coverslip out of PBS, drain out the excess fluid quickly by putting the edge of the coverslip on kimwipe and mount it on the slide with the cell side facing the Vecta Shield
15)   Carefully aspirate off the excess Vectashield coming out from the side of the coverslip without moving the coverslip (If unsure omit this step). Seal the edges of the coverslip with clear nail polish.
16)    Once the nail polish is dry the coverslip can be used for imaging.

Re: Cells detachment after fixation
« Reply #1 on: September 08, 2009, 04:58:29 PM »