PROBLEM:
Visible on the oscilloscope
POSSIBLE CAUSES:
- From damaged electrode/non-tight connection
- From vibration of perfusion
- From grounding error
First, check the noise can be seen in all channel or not. If not, the noise is caused by either bad electrode or bad connection between connector and probe. Then, check with static condition. If noise happens in the static condition, the noise should come from grounding error. If not, the noise should come from vibration of perfusion.
SOLUTIONS:
From damaged electrode / non-tight connection
Turn the direction of probe. If noise channel would move, the noise is caused by electrode. Please use new one. If noise channel would not move, the noise is caused by bad connection. Wipe the terminals of both probe and connector with ethanol, then push connector pins on connector without direct touch (e.g. use globe or lint-free paper) several times to recover good contact.
From vibration of perfusion
Check the ground connection of inlet tube of perfusion cap, if the noise has 50/60Hz cycle. If not, re-adjust vacuum pressure or speed of peristaric pump
From grounding error
Check the grounding from cable of connector. If the connection is secure, wrap the entire connector with aluminum foil which is connected to the ground.
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PROBLEM:
Unable to maintain
SOLUTION:
The vacuum pressure is too high/low. Adjust to ~50-70 psi (negative pressure).
The perfusion outlet tube is damaged/clogged. Therefore, replace or clean.
Replace gravity/vacuum system to peristaric pump.
PROBLEM:
Too high
SOLUTION:
Re-adjust the height of the perfusion outlet.
PROBLEM:
Too dry
SOLUTION:
Re-adjust the height of the perfusion outlet.
QUESTION:
What is interface?
ANSWER:
This is defined as when the solution level is visibly moving over the tissue sample. Mainly noticed with a slight increase of the solution level within the region of the tissue.
QUESTION:
What is submerged?
ANSWER:
This is is defined with the level of solution completely submerging the tissue sample.
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PROBLEM:
Unable to see a response
SOLUTIONS:
For simple EPSP responses in hippocampus
Prep your slices with a narrow cutting angle (30 degrees) target region of the hippocampus: mid to dorsal.
For simple rhythmic activity in hippocampus
Prep your slices with a steeper angle of or near 40 degrees. Target region: dorsal region.
Other tips:
Poor adhesion of the tissue and the electrode will result in no response. Readjust the tissue and/or replace the tissue.
The pressure from the net and the weight may potentially damage the tissue. Therefore, decrease the weight by using a smaller piece of netting. Vice a versa if the pressure is too low.
Decrease the level of solution in MED probe.
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PROBLEM:
Hard to believe the response
SOLUTIONS:
C heck various sites of stimulation, especially across the cell body layer. If you observe the reversal effect across the cell body, then your response is a true response. However, if you don't see this effect, then you have poor adhesion between the tissue and the electrodes.
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PROBLEM:
Observed a sudden decrease in the response
SOLUTIONS:
- A high perfusion pressure can cause the slice to shift slightly.
- A change in temperature can cause this change
- Poor perfusion technique, ie. Using a 3-way value to switch from and into different solutions within your protocol.
- An air-bubble can potentially disrupt the stable environment , and thus suddenly change the response.
- The decrease in the O2 concentration in the solution can cause an change in the response.
- The pressure from the net and the weigh may be overwhelming to the sensitive tissue. Decrease the weight by cutting out a smaller piece of the net.
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PROBLEM:
In the hippocampal slice, the region of interest is too narrow, too small, or too large.
SOLUTIONS:
Check the angle of your dissection. And also, monitor which portion of the hippocampus you are using, ventral to dorsal and study which section will be more dominant.
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PROBLEM:
Vibration caused by the building, high traffic, and touching/bumping
SOLUTIONS:
Place vibration absorbers under the legs of all the apparatuses in your setup (table, incubator).
