How to prepare acute hippocampal tablets of rats
The hippocampus is a classic brain region that studies learning and memory and plays an important role in declarative memory.
Although now in vivo electrophysiology and in vivo imaging technology plays more and more important role in behavior, classic in vitro hippocampal slices of electrophysiological technique and imaging technology, still has an irreplaceable position in the field of neuroscience.
For example, the multi-cell membrane forceps of the isolated hippocampal brain tablets may be used to study the neural microloop.
At present, in the body membrane forceps with single cell diaphragm forceps, in the body multi-channel is limited to local potential records, it is difficult to achieve multiple cells in the body diaphragm pliers.
In addition, in vivo imaging technology, or can only reach the level of cell resolution (e.g., calcium in vivo imaging), or can only watch a single goal at a time (such as in vivo two-photon dendrites of observation, because multiple two-photon laser is not you want to buy can buy).
And the use of hippocampal slices, we can easily on the confocal microscope, real-time imaging at the same time for multiple fluorescent protein (ordinary laser very cheap), cell bodies and dendrites can be easily and imaging.
If you combine the patch clamp and the image together, it's definitely a wise choice to leave the hippocampus, which is easy and cheap.
Add a new neuro-scientific new light genetic technique, and that's what happens in minutes on the brain.
It's not impossible to combine all three, but it's much harder, and the cells that can record and observe are very limited.
So much to say, the students of neuroscience are to prepare this ancient, simple and practical technique for the preparation of acute hippocampal brain tablets.
Acute , the emphasis is on speed, of course, on the premise of guaranteeing quality.
Unlike in the body experiment, the mice did not have any effect for several hours.
In vitro, it is best to finish in 10 minutes, not more than 15 minutes.
The importance of ensuring the activity and function of brain cells does not need to be emphasized.
In addition to speed, the scientists have also worked hard on Artificial cerebrospinal fluid (ACSF), such as Gey's solution, Ringer solution, and the secret recipe of the old Mr. Fung (DOI: 10.1007/978-1-4 939-1099-0_14).
The experiment to prepare
1, crushed ice,
2. Artificial cerebrospinal fluid: pour into a beaker, cool in ice water mixture, and pass 95% O2/5% CO2
3. Vibration slicer (such as Laica VT1200 S), blade
4. Water bath pan
5. Knife, eye scissors, biting forceps, curved hook, spoon, scalpel, blade, filter paper, glue, syringe 2, straw, small beaker, as shown below:
The solution configuration
Slice liquid (in mM) : 92 NaCl, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 25 Glucose, 20 HEPES, 3 Na + - pyruvate, 10 MgSO4, 0.5 CaCl2;
Use deionized water.
Artificial cerebrospinal fluid (in mM) : before each experiment, 10XACSF was diluted 10 times, and the group was divided into: 124 NaCl, 2.5 KCl, 2.5 cacl 2, 2 MgCl2 • 6H2O, and 1.25 KH2PO4 of 1X artificial cerebrospinal fluid.
Add sodium bicarbonate and glucose to 1 * artificial cerebrospinal fluid, and the final concentration is 26 mM NaHCO3 and 10 mM.
Section 1 x, liquid 4 ℃ preservation;
Artificial cerebrospinal fluid 10 x, 4 ℃.
The experimental process
Prepare for
The artificial cerebrospinal fluid was precooled in ice water mixture for at least 20 minutes, with 95% O2/5% CO2 (4 L/h).
Precool the surgical instruments in a mixture of ice water
Take the brain
1. Anesthetized rats, the neck of the rat is placed on the knife, quickly severed head.
Roll up the scalp, reveal the skull, and use the scissors to extend the center of the skull to the Bregma, and then cut a knife in the position of the two sides of the ear
2. Remove both sides of the skull from the caudal side with the bite forceps
3. Use the crochet to pick out the brain and cool 2 min in the pre-cooled artificial cerebrospinal fluid
Fix the brain
4. Drip two drops of powerful glue on the platform, spread evenly, making the glue area slightly bigger than the paste area of the brain.
5. Scoop out the brain with a spoon and place it in the slice slot and back up
6. Use a knife to fix your brain.
Cut to the cerebellum.
Will cut the brain for about two and a half the brain, then turned half a brain, make plane (Medial) down, then half back side of the brain (dorsal) to cut to about a quarter of 70 °
7. Use a flat shovel to transfer the half brain to the filter paper and suck out the excess moisture
8. Place the side of the side of the half brain on the glue of the platform, and put it in a figure of eight
slice
9. Place the platform in the slice slot, pour into the pre-cooled artificial cerebrospinal fluid, and continue to pass the oxygen (2 L/h). If the oxygen is too large, it can stir the cerebrospinal fluid, so that the brain can spin and float
10. Adjust the blade activity interval of the slicer, starting at 3 ~ 5 mm from the brain, and stop the position of the intact seahorse.
In addition to ensuring that the intact hippocampus is cut, the slicer can be avoided and the sliced time is shortened
11. Bring the platform to the right height.
This suitable height will be the first to reveal the complete structure of the hippocampus.
This should be summarized by the experimenter.
Rise to the appropriate height, also to shorten the slice time, avoid the slicing machine to do the work
12. Start the slice according to the start button of the slicing machine. When the whole hippocampal tablet comes out, the hippocampus slice will be cut off with a syringe needle
13. Remove the excess cortex from the hippocampus with a syringe
Incubation
14, hippocampal slices through a straw will be transferred to more than 30 ° C artificial cerebrospinal fluid incubation for 1 hour.
95% O2/5% CO2 (4 L/h)
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