ACSF

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(New page: '''Artificial Cerebrospinal Fluid (ACSF)''' is a isotonic, glucose-rich buffer used when working with organotypic neural preparations. A mixture of 5% CO<sub>2</sub> / 95% O<sub>2</sub> is...)
Current revision (13:01, 15 January 2009) (view source)
 
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#Should be approximately 310 mOsm and pH 7.4 when bubbled with O<sub>2</sub>
#Should be approximately 310 mOsm and pH 7.4 when bubbled with O<sub>2</sub>
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Uses:
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'''Uses:'''
This buffer is often used during vibratome sectioning of a brain for organotypic slices. Prepare a 4% low-melting point agarose solution in ASCF, by placing LMP agarose into the appropriate amount of ACSF and microwaving it until dissolved. One must watch it while it heats, as it boils over very easily. Starting and stopping many times throughout the heating is common. Then place the 4% LMP-agarose/ACSF in a 37°C water bath to cool off to just over the temperature where it solidifies (this is to prevent heat shocking the brain you're putting into it). When the solution reaches temperature, pour into a small petri dish and then quickly place your brain into it in the orientation desired. Then place the petri dish (or whatever receptacle suites you) on ice while the agarose sets.  
This buffer is often used during vibratome sectioning of a brain for organotypic slices. Prepare a 4% low-melting point agarose solution in ASCF, by placing LMP agarose into the appropriate amount of ACSF and microwaving it until dissolved. One must watch it while it heats, as it boils over very easily. Starting and stopping many times throughout the heating is common. Then place the 4% LMP-agarose/ACSF in a 37°C water bath to cool off to just over the temperature where it solidifies (this is to prevent heat shocking the brain you're putting into it). When the solution reaches temperature, pour into a small petri dish and then quickly place your brain into it in the orientation desired. Then place the petri dish (or whatever receptacle suites you) on ice while the agarose sets.  
A block of agarose containing the brain can then be cut out of the agarose prep and affixed with super glue to the bottom of a glass dish for vibratome cutting. Fill the dish with oxygenated, 4°C ACSF and section to the desired thickness. Immediately transfer these sections to a container filled with ASCF with 5%/95% CO<sub>2</sub>/O<sub>2</sub> gas mixture bubbling through it.
A block of agarose containing the brain can then be cut out of the agarose prep and affixed with super glue to the bottom of a glass dish for vibratome cutting. Fill the dish with oxygenated, 4°C ACSF and section to the desired thickness. Immediately transfer these sections to a container filled with ASCF with 5%/95% CO<sub>2</sub>/O<sub>2</sub> gas mixture bubbling through it.

Current revision

Artificial Cerebrospinal Fluid (ACSF) is a isotonic, glucose-rich buffer used when working with organotypic neural preparations. A mixture of 5% CO2 / 95% O2 is bubbled into it through an airstone, while it is cooled in an icebath for at least 10 minutes before use with a brain.

10x ACSF					
		        Mw (g/mol)	1x (mM)	  10x (M)	10x (g/L)	100mL (g)
R1	NaCl	        58.44	        125	  1.25	        73.05	        7.31
	KCl	        74.55	        2.5	  0.025	        1.86	        0.186
	MgCl2*6H20	203.3	        1	  0.01	        2.033	        0.203
	CaCl2*2H20	147	        2	  0.02	        2.94	        0.294
	NaH2PO4	        138             1.25      0.0125        1.725	 
	 	 	 	 	 	 
	 	 	 	 	 	 
R2	NaHC03	84.01	25	0.25	21.0025	2.1
	glucose	180.2	25	0.025	4.505	0.451 
  1. Sterile filter R1 & R2 and store at 4°C
  2. 100mL R1 + 100mL + 4.5g glucose + 800 H20
  3. Should be approximately 310 mOsm and pH 7.4 when bubbled with O2

Uses:

This buffer is often used during vibratome sectioning of a brain for organotypic slices. Prepare a 4% low-melting point agarose solution in ASCF, by placing LMP agarose into the appropriate amount of ACSF and microwaving it until dissolved. One must watch it while it heats, as it boils over very easily. Starting and stopping many times throughout the heating is common. Then place the 4% LMP-agarose/ACSF in a 37°C water bath to cool off to just over the temperature where it solidifies (this is to prevent heat shocking the brain you're putting into it). When the solution reaches temperature, pour into a small petri dish and then quickly place your brain into it in the orientation desired. Then place the petri dish (or whatever receptacle suites you) on ice while the agarose sets.

A block of agarose containing the brain can then be cut out of the agarose prep and affixed with super glue to the bottom of a glass dish for vibratome cutting. Fill the dish with oxygenated, 4°C ACSF and section to the desired thickness. Immediately transfer these sections to a container filled with ASCF with 5%/95% CO2/O2 gas mixture bubbling through it.

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