I am currently working on setting up primary neuron culture in the lab. I use E18 Rat or E16.5 Mouse. I use HBSS supplemented with HEPES and P/S for dissecting buffer. I use a Complete Neurobasal medium supplemented with B27 and Glutamax to culture neurons. I am trying to see the morphology of neurons at DIV2. I plate 100,000/well neurons (for Hippocampal neurons) on the 18mm-coverslip (1mg/ml PLL-coated) in a 12-well plate. The problem is whenever I do the ICC, they look swollen when I detect using Phalloidin. My neighbor lab is also doing neuron culture but whenever I use their coverslip-cultured neurons, it looks totally fine (thin). I use exactly all the same buffer as my neighbor lab. Also, I tried to use their neurons dissected from them and I plated neurons on my coverslips. But, they also look swollen after ICC. I used exactly the same ICC protocol for both my coverslip-cultured neurons and their coverslip-cultured neurons. So, I think there should be some difference in the culturing step after dissection and before ICC.
Btw, they both look totally fine when I observe them using a dissection microscope until I do the ICC and acquire Image using a Confocal microscope.
Did you use exactly the same Staining buffers?? Depentent on the ion concentration of the PBS there could occur differences....
Hi Sung, since you are performing a primary neuron culture, there will be cells that are dissected from the hippocampus including glia that can grow in your wells along side neurons. Glia can often grow extremely rapidly and take over the neurons, as such we supplement our primary hippocampal neurons with FUDR at 10uM final concentration at 2DIV~7DIV to prevent glial overgrowth (our plating medium contains 1% FCS, as we find this increases the longevity of our neurons, but inadvertently provides a better environment for glial survival) .
My guess is that your neurons aren't swelling - in your first image I can clearly see a pyramidal-like cell with nice processes, but rather, your wells have a greater proportion of glia which naturally have this "swollen" morphology in culture. Try staining for a glial-marker and test if this is the case. If so, potentially supplement your wells with FUDR after plating (note, supplementing wells with FUDR can be toxic for your neurons).
Your ICC looks great!