First, thanks for all the folks here posting problems and anwering questions, I've learned a lot from here.
Recently I met a problem,
In my experiment, I stimulate schaffer collateral fibers and record evoked EPSCs in CA1 pyramidal neurons using whole-cell recording in mice hippocampus.
When I was using low stimulus strengh (~0.1mA), EPSCs looks normal, However, If I stimulate with a relatively high stimulus strength (~0.5mA), sometimes I will get a fast inward current spike at the peak of EPSC (with K-gluconate intracellular solution, see attached picture) and get prolonged large EPSCs (with Cs+ based intracellular solution, EPSC last for several hundred of miliseconds, the shape is definitely not normal), and I guess that's a result of losing voltage control over distal dentrites when the deporlarizing current was very big. So I put QX-314 in my intracellular solution to block Voltage-gated Na+ channels intracelluarly. After putting QX314, I saw much less prolonged EPSCs at first. However, after about a month, the prolonged EPSCs appear again...And I have almost no idea what's causing the "fat EPSCs" except worrying about the degradation of QX-314)
My questions are:
1, what concentration of QX-314 you guys put in intracellular solution when recording large EPSCs?(I am using 5mM)
2, QX-314 is said to be light sensitive. so I made the solution in dark condition and wrap my intracellular solution syringe with foil during recording) Since my recordings usually last for 1hour, Is that necessary to cover the whole rig with some black cloth to avoid light?
3, On MSDS sheet of QX-314, it is said that the stock solution should be used within one month. So, should I make intracellular solution with new stock of QX-314 every month? (I use to make a huge amount of intracelluar solution stock which is good for 3 months and put it in -80 freezer, have any of you tried store intracellular solution with QX-314 more than a month?)
Any other advices on handling QX-314 or getting better voltage control are welcome!!