By 'after D1' do you mean after D1 of differentiation, or fixation?

Some cells detach easily when you fix and wash with PBS so I'd be careful of cell density (~80% but not too full), and be gentle during your liquid handling.

Otherwise, there might be some issues with your differentiation protocol than immunofluorescence if you can't get enough cells in the first place.

I meant after Day 1 Of starting IF staining.

Initially, we seeded the cells at 75% confluency. However, they grew in size & number then differentiated into neurons. They formed a network of processes and connected neurons. At day 14 after differentiation, we started the IF staining protocol.

So I assume 'Day 1' of immunofluorescence/fixation.

I would be careful with your washes. I have had cells come off the wells in IF. Maybe also use a pre-coated plate or coat it yourself with poly-D-lysine.

Leran Mao I used precoated Ibidis 96 well plate. Could 4% PFA not sufficient to fix neurons? Could we use another reagent with it?

4% PFA is usually good. You can also try methanol. Fixing at a lower temperature might also help. If you fixed for 24 hours, you can try shortening your incubation time. E.g. 1 hour at RT - I'm not sure of a typical condition for neurons.

But the point is the next time take more care with any liquid handling during washes. The fact that the cells are fixed does not mean they will not collectively come off the plate.

Actually, we do washing only after adding primary & secondary antibodies. Only 3 times each that last 3 min.

Then, one last wash with PBST.

Lastly, we wash with water.

then we add Hoechst for 10 min.

Do 1 wash with PBS.

then we add PBS to the wells. Then we do imaging.

In each time, we add slowly towards the wall.

Have you tracked cell density in any of your incubations? Cells could be washed away in any steps if your liquid handling is harsh.

I also edited my previous comment to suggest a shorter incubation time. Incubating for a long time could rigidify the cells and likely make them detach easier. I usually incubate for

I would recommend you optimize your fixation condition (10 min - 1 hr, RT/4C) and monitor cell density on a microscope throughout your washes to determine which steps result in loss of cells.

Leran Mao We only do fixation with 4%PFA for 10 min only.

We do IF staining in two days with incubation with primary antibody overnight. I took a look at day 2, I found 2 wells were empty & 1 well has the neurons drift away.

At the end of Day 2 of IF staining, I lost my neurons in 7 out of 8 wells.

Hi Hanadi Marzouq,

I agree with all of what Leran Mao said, but wanted to stress some of the things. I also work with neurons, so I know they can be tricky. Please check exactly how your plates are precoated. Often, this is enough for cells like fibroblasts or HeLa cells, but neurons (and especially neurites) detach easily and need better/additional coating. Like mentioned, you could try poly-d-lysine, but there are many options out there including laminin or also matrigel or geltrex (mainly for human cells).

Then you need to be as gentle as possible while removing and adding any liquids to the wells. Do not use any vacuum pumps as they are too strong.

10 min of fixation with 4% PFA is perfectly acceptible. One option would be to add 8% PFA directly into the cell culture medium in a 1:1 (vol/vol) dilution. That way you can reduce the number of pipetting steps.

It is great that your neurons form dense networks, but unfortunately that also means that they come off as the whole network, so the key really is good attachement to the bottom of the wells and gentle pipetting.

I hope this helps.

Best of luck,

Selene