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127 mM NaCl
1.0 mM KCl
1.2 mM KH2PO4
26 mM NaHCO3
10 mM D-glucose
Weigh out salts using a fine balance to make up 2 L of artificial cerebrospinal fluid (aCSF) solution. Top it up to with deionised H20 to around 1.9 L, leaving enough room to add other components later. Shake well and make sure all salts have dissolved. Bubble aCSF with carbogen 95% O2/5% CO2 for 15-20 minutes to stabilize the pH to 7.3-7.4. Afterwards add 4.8 ml of 1M CaCl2 and 2.6 ml of 1M MgCl2 using a suitable pipette to give a final concentration of:
2.4 mM CaCl2
1.3 mM MgCl2
Top it up with deionised H20 to 2 L. Bubble for a further 15 minutes with carbogen.
140 mM Potassium gluconate
10 mM KCl
1 mM EGTA-Na
10 mM HEPES
4 mM Na2ATP
0.3 mM Na2GTP
pH adjusted to 7.3 with 0.5M KOH and the osmolarity adjusted with 1M sucrose.
Tips: It is important to continually top up the chamber with cold aCSF solution to keep the brain tissue cool whilst slicing.
If unfamiliar with brain anatomy, use a reference such as Paxinos and Watson, The Rat Brain Atlas to ensure slicing of the correct brain area.
Image: Figure 35 from the Rat Brain Atlas
Tip: It is important to ensure the batch specific molecular weight is used to determine an accurate concentration of test compounds. Check out our FAQ on small molecules for more information.
View our IHC protocol.
The video protocol was produced by Abcam in partnership with NeuroSolutions.
Weigh out salts using a fine balance to make up two liters of artificial cerebrospinal fluid solution, aCSF. Top it up with deionized water to around 1.9 liters, leaving enough room to add other components later. Shake well and make sure all salts have dissolved.
Bubble aCSF with carbogen, 95% oxygen, 5% carbon dioxide for 15 to 20 minutes to stabilize the pH to 7.3 to 7.4.
Afterwards add 4.8 mils of one molar calcium chloride, and 2.6 mils of one molar magnesium chloride, using a suitable PET to get a final concentration of 2.4 millimolar and 1.3 millimolar.
Top it up with deionized water to two liters. Bubble for further 15 minutes with carbogen.
Then chill one liter of aCSF over ice until it is cold at or below four degrees C.
Mount a double-edged razor blade onto the knife holder. Screw to tighten into place.
Set the microtome to the desired thickness. Two to 500 microns for brain slices and adjust the cutting speed to the desired setting. In this case, number three or nought 0.15 millimeters per minute.
Place the brain into pre-chilled aCSF solution.
For hippocampus sections, trim the brain by cutting off the cerebellum, which provides a flat surface to mount the brain with and a small part of the prefrontal cortex.
Mount the brain, cerebellum side onto the microtome specimen disc using super glue.
Orienting the sample such that the cortex faces the razor blade.
Add a supporting piece of agar behind the brain away from the slide of the viva tome to provide structural support during the slicing.
Set the limit stop position to the microtome to define the start and stop positions of the slicing.
Set the frequency of the microtome to maximum, 100 hertz.
Use the up-rocker button to move the buffer tray and brain to a position where the exposed surface is just below the razor blade edge and press start to begin the brain slicing.
It is important to continually top up the chamber with cold aCSF solution to keep the brain tissue cool while slicing. If unfamiliar with brain anatomy, use a reference such as [plaxine 00:03:24] or [Watson 00:03:25] the map brain atlas to ensure correct slicing of a brain area.
Easier transfer for PET, transfer each individual brain slice containing the region of interest from the buffer tray to a clean Petri dish prefilled with chilled aCSF.
Carefully hemisect the brain down the midline and transfer the individual slices to a holding chamber prefilled with aCSF.
Stool the slices for at least one hour at room temperature to allow the brain tissue to recover from the mechanical shock of slicing.
Prepare intracellular recording solution into a one mil [epindorph 00:04:06] tube from pre-prepared stock solutions.
Here, we added Alexa-633 dye to a final concentration of 50 micromolar to allow for further IHC experiments of the recorded brain neurons.
Make an electrode filler my melting a one mil plastic pasture for PET over a Bunsen burner flame. Once the pasture for PET has turned opaque within the flame, peel each end apart to stretch the plastic and once cooled down, cut allowing enough length to reach the bottom of the recording for PET.
Fill the electrode filler with the intracellular solution.
Fabricate a glass recording for PET by using appropriate glass capillaries and perpet puller.
It is essential to ensure that the batch specific molecular weight is used to determine an accurate concentration of test compounds.
On all our chem compounds, the batch specific molecular weight is clearly displayed on the vile.
Prepare the test compound and make the stock solution by weighing out the compound by using a fine balance.
Stabilization instructions can be found on the certificate of authenticity accompanying your product or on the website.
Some products may be difficult to solubilize, and you may find that rapid stirring, warming in a water bath or [sonication 00:05:26] of the solution may help.
Solubility is temperature dependent. As such, cooling or freezing solutions may lead to precipitation of a product after solution. It is therefore important to ensure that your product is completely redissolved before use. After making up the stock solution, [inaudible 00:05:45] at the desired volume and freeze until use.
On the day of experimentation, defrost a single tube and add it to the aCSF to achieve the final working concentration that will be used in the experiment.
Now, the solutions have all been produced, the recording can start. Fill up a bottle with aCSF solution and testing compound solution.
Bubble both with carbogen and adjust the flow rate of the aCSF solution to approximately five mils per minute using metal Hoffman clamps.
After one hour incubation and room temperature, carefully place the brain slice into the recording chamber using a transfer perpet or small brush. Top up the chamber with aCSF solution. Move the slice into place using a small brush and secure the slice with reference electrode.
Fill the glass recording perpet with intracellular solution using the perpet filler, making sure the solution is all the way down at the tip of the perpet. Flick to get rid of any air bubbles. Attach the perpets to the electrode holders with a patch clamp amplifier head stages and turn into position.
Using fine control micro manipulators, descend the recording perpet to the region of interest within the brain slice. Here, the CA1 region of the hippocampus.
If required, use a course manipulator top position an appropriate stimulating electrode again to the appropriate region of the brain slice to stimulate inputs to recorded neurons. Here, this is within the shaft of collaterals of the hippocampus.
Use the control dials of the micro manipulator to descend the recording perpet into the aCSF solution covering the brain slice. Open the seal test function of the computer controlled acquisition software and monitor the resistance of the recording perpet.
The ideal resistance when filled with intracellular recording solution is between five and eight mega ohms.
Using the perpet offset or track functions of the recording amplifier, set the offset current to zero prior to advancing the electrode into the brain slices with the micro manipulator.
At the same time, apply pressure to the recording electrode via a syringe connected to the electrode holder. When a neuron is encountered, the conductivity through the recording electrode is increasingly reduced as the tip of the recording perpet gets closer to the neuronal membrane.
Gentle positive pressure is applied via the syringe to clear the tip of the recording perpet of any debris that may have occluded as the recording electrode is lowered through the brain slice.
Once close to a neuron, the height of the injected current step will have fallen to approximately 20% of its starting value.
Negative pressure is then applied then via the syringe to obtain a giga ohm seal and negative current applied to the electrode via the acquisition software.
After allowing the giga ohm seal to improve over time, we apply further negative pressure via the syringe to rupture the membrane and gain direct access to the cytoplasm of the neuron whilst maintaining a tight seal to prevent current leakage. We now have wholesale access to the recorded neuron as defined by the increase capacitance transience visible in our current step.
Using the patch clamp amplifier, the capacitance transience are progressively canceled and electrophysiological experiments can begin on the recorded neuron. Using the computer controlled acquisition software, the health and electrophysiological properties of the recorded neuron are assessed. A current voltage relationship is constructed by injecting square wave negative and positive rectangular current injection of constant increment into the neuron.
This move membrane trace, an action potentially firing on the positive current steps, indicates the health of the recorded neuron. In the current clamp recording mode of the wholesale patch clamp technique, the neuronal membrane responses to apply test compounds can be assessed.
In order to test the effects of test compounds, switch the recording solution from aCSF in the main aCSF bottle to the pre-prepared test compound contained within 50 mil syringes connected to the recording chamber by three-way taps.
In this example, we are applying a one micro molar [trotodo 00:10:55] toxin a potent [sadium 00:10:57] channel blocker to a recorded neuron in the current clamp recording mode of the wholesale patch clamp technique. The cessation of spontaneous action potential firing can clearly be observed.
Using the stimulating electrode placed within the shaft of collateral pathway of the hippocampus, the responses to electro clear volt post synaptic potentials can also be monitored. Here, a mixed [inaudible 00:11:21] glutamatergic EPSP, an inhibitory gabaergic IPSP can be seen in response to electrical stimulation.
Once the experiment has finished, the brain slice is removed from the recording chamber using a paint brush and carefully placed within a suitable glass container.
A perpet is used to fill the container with 4% pure formaldehyde and 0.1 molar phosphate buffer, pH 7.4 and stored a fridge until used for immunized to chemical processing.