Bubbles in the tissue

shao

After ETC, there are several bubbles in the tissue, why and how to fight down it?

Fabio

For me the best way to avoid bubbles is to play with the flow-rate of the buffer.
The bubbles result from the electrolysis of water.

shao

I'm sorry, I means that the bubbles in the brain at the end of the ETC, not means in the ETC chamber or tube.

Kristin_Engberg

Keeping a flow of the clearing solution in the chamber throughout ETC is essential to preventing bubble accumulation in the brain sample. If you have no flow, the bubbles created during ETC may become trapped in the sample. Even with flow, you may need to adjust the rate to prevent a large accumulation of bubbles in the top of the chamber that may not be completely separated from your sample.

raju

Just to add, bubbles inside the tissue might also be indicative of damage to the tissue, possibly due to high voltage or low flow rate. What voltage are you applying Shao?

shao

Voltage: 30 V; Temperature: 37 ºC; Flow rate: 50 ml/min; Current: changed from lower to higher (1.6-1.8 A).

nicolasrenier

I also have bubbles trapped in the ventricules in adult brains. Those are really large (the size of the ventricule, basically). I also use a very high flow rate : the max allowed by the Lauda water bath, a couple of Liters per min.

If I remove the cortex to expose the thalamus, I don't have any bubble. Maybe the bubbles form in the ventricular space, and are too big to be evacuated?

raju

Shao, 50 ml/min is too little for 30 V (assuming standard spacing between the electrodes). This flow rate will not dissipate the heat fast enough. I suspect the bubble could comes from over-heating. 2-3 Liter/min will be better, or go down in voltage - 10V or so, or go down in temperature: 15-20 deg C.
How much voltage and electrode separation are you using Nicolas?

nicolasrenier

raju said:

How much voltage and electrode separation are you using Nicolas?

I use the design from the paper for the chamber, and run at 30V. It is possible that even at this voltage, the system is overheating. I'll try 20V next time! Also, I have a new water bath with a cooling block, which should prevent the temp from ramping up in the circuit.

More tests coming up!

raju

For the temperature part: SDS micelle formation is dependent on temperature (and SDS concentration). Check out this recent paper: "Temperature Effect on the Nanostructure of SDS Micelles in Water" http://nvlpubs.nist.gov/nistpubs/jres/118/jres.118.008.pdf all the figures 6,7,8,9,10 are very informative. Figure 6,7 are temperature vs micelle size. Size goes up by lowering the temperature, implying harder for micelles to get out of pores at lower temp. So, lowering the temperature is probably not an alternative for low flow rate.
Same for the SDS concentration.