Brain Slice Chamber System User`s Manual
Contents
1. be purchased at a fabric store A wide mouthed pipette made by cutting the tapered end off a Pasteur pipette can be used to transfer slices to the chamber This procedure should be done with the solution flow stopped When transferring slices a fine bristled brush should be posi tioned between the mouth of the pipette and the end of the mesh nearer to the out flow dam The brush can then be used to position the slice Immediately before start ing the solution flowing place a few drops of the perfusing solution on the slice so that the chamber is filled This will prevent surface tension from lifting the slice a result when the chamber fills from the side A flow rate of 1 ml min may suffice at room temperature while warmer temperatures will require higher flow rates To insure even flow through the chamber a nylon mesh similar to the mesh supporting the slice can be positioned between the inflow port and the support mesh A newly cleaned chamber may slurp that is fluid will alternately build up then flow over the dam If this occurs place a piece of tissue paper or cotton thread at one edge of the dam to act as a wick Step ZT 3 The investigator is to supply the necessary electrical ground The ground device agar bridge or Ag AgCl wire should be positioned between the end of the mesh and the outflow dam to prevent disruption of fluid flow over the slice Be aware that the posi tion of the ground may effect the fluid level near the dam2. for the remainder of the original warranty period dating from the purchase of the orig inal device This warranty gives you specific rights and you may also have other rights which vary from state to state Repair Facilities and Parts Harvard Apparatus stocks replacement and repair parts When ordering please describe parts as completely as possible preferably using our part numbers If practi cal enclose a sample or drawing We offer a complete reconditioning service A CAUTION Not for clinical use on human patients Publication 5407 001 REV D www harvardapparatus com 4 Harvard Apparatus Brain Slice Chamber System User s Manual Introduction Congratulations You have purchased one of the finest Brain Slice Chambers available to aid you in your research You will find the Harvard Apparatus Inc unit easy to use and easy to maintain The Harvard Apparatus Inc brain slice chamber systems offer the investigator two methods of maintaining thin slice of brain tissue in vitro When used in conjunction with a common base unit BSC BU either the BSC HT Haas Top or the BSC ZT Zbicz Top will maintain brain slice preparations for many hours and allow for stable intra cellular or extracellular recording The BSC ZT is a full submersion chamber in which solutions flow transversely to the cut surfaces of the slice The BSC HT is an interface type chamber in which one cut surface of the slice is exposed to a humidified gas mix tur
3. the unit s Warranty Harvard Apparatus warranties the instrument s for a period of one year from date of purchase At its option Harvard Apparatus will repair or replace the unit s if it is found to be defective as to workmanship or mate rial This warranty does not extend to damage resulting from misuse neglect or abuse nor mal wear and tear or accident This warranty extends only to the original customer purchaser IN NO EVENT SHALL HARVARD APPARATUS BE LIABLE FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES Some states do not allow exclusion or limitation of incidental or consequential damages so the above limitation or exclusion may not apply to you THERE ARE NO IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR USE OR OF ANY OTHER NATURE Some states do not allow this limitation on an implied warranty so the above limitation may not apply to you If a defect arises within the one year warranty period promptly contact Harvard Apparatus Inc 84 October Hill Road Holliston Massachusetts 01746 1371 using our U S only toll free number 1 800 272 2775 or dial 508 893 8999 Goods will not be accepted for return unless an RMA returned materials authorization number has been issued by our customer service department The customer is responsible for shipping charges Please allow a reasonable period of time for completion of repairs replacement and return If the unit is replaced the replacement unit is covered only
4. top unit is machined to fit into the round opening at the top of the base unit it is recommended that dental wax or some easily removed adhe sive e g rubber cement be used to secure the top unit In addition two screw holes and two screws have been provided to secure the BSC ZT to the base unit Publication 5407 001 REV D www harvardapparatus com 4 Harvard Apparatus Brain Slice Chamber System User s Manual BSC ZT Zbicz Top Step ZT 2 An opening has been drilled through the side of the top unit Fig 3b for placement of a thermistor probe BSC T2 near the outflow hole at the end of the trough The thermistor probe is inserted through the luer fitting at the side of the top unit and posi tioned so the thermistor is in the middle of the trough Slices in the BSC ZT can be held on the mesh by a number of methods As the per fusing solution flows transversely there is little tendency for slices to move vertically Therefore slices can be held at a few points that allow for stable recording with micro electrodes Slices can be held to the mesh with stimulating electrodes the plastic insert Fig 3c or other wire holders The number of points at which a slice is immo bilized depends on the size of the tissue For example guinea pig hippocampus approximately 3 x 6 mm is stable when held at 4 points while smaller slices may require only 2 or 3 points Slices can also be held by covering them with a piece of fine nylon net which can
5. 202A The electrical connection from the Base Unit to the Temperature Controller is made by an I O cable which has a 6 pin din connector at one end and 2 banana plugs Red and Black plus a Ring Tongue for Chassis Ground at the other end A way to minimize 60Hz noise is by referencing your recording or measuring equip ment to the shield of the BNC in the front panel of the TC 202A Figure 2a TC 324B Temperature Controller The heating element is connected to two banana jacks connectors on the side of the base unit Fig 1a A special version of the Base Unit BSC BUW 64 1525 maybe used with the Warner TC 324B TC 344B single and dual channel Temperature Controllers Haas Top or Zbicz Top Publication 5407 001 REV D www harvardapparatus com Harvard Apparatus Brain Slice Chamber System User s Manual BSC ZT Zbicz Top 3c 3b 3a Figure 3 The BSC ZT Zbicz Top The BSC ZT Figure 3 is a submersion type chamber in which brain slices are sup ported on a stiff nylon mesh to allow the perfusing solution to flow transversely across both cut surfaces Step ZT 1 To assemble the slice chamber using the BSC ZT position the top unit so that one of the clear plastic tubes protruding through the top of the base unit Fig 1e or 1f can be connected to the fitting on the bottom of the BSC ZT Fig 3a and the top can be centered on the BSC BU After this connection has been made place the top unit onto the base unit Although the
6. Brain Slice Chamber System User s Manual BSC BU Brain Slice Chamber System Base Unit MAI 65 0073 BSG ZT Brain Slice Chamber System Zbicz Top MAI 65 0074 BSC HT Brain Slice Chamber System Haas Top MAI 65 0075 BSC PC Brain Slice Chamber System Prechamber MAI 65 0076 BSC BUW Brain Slice Chamber Base Unit MAI 64 1525 HARVARD APPARATUS Publication 5407 001 REV D WEEE RoHS Compliance Statement EU Directives WEEE and RoHS To Our Valued Customers We are committed to being a good corporate citizen As part of that commitment we strive to maintain an environmentally conscious manufacturing operation The European Union EU has enacted two Directives the first on product recycling Waste Electrical and Electronic Equipment WEEE and the second limiting the use of certain substances Restriction on the use of Hazardous Substances RoHS Over time these Directives will be implemented in the national laws of each EU Member State Once the final national regulations have been put into place recycling will be offered for our products which are within the scope of the WEEE Directive Products falling under the scope of the WEEE Directive available for sale after August 13 2005 will be identified with a wheelie bin symbol Two Categories of products covered by the WEEE Directive are currently exempt from the RoHS Directive Category 8 medical devices with the exception of implanted or infected products and Category 9 mon
7. Harvard Apparatus Brain Slice Chamber System User s Manual Cleaning Instructions Run distilled water through the chamber and perfusion lines after every use Water should not be allowed to remain in the perfusion lines or chamber as this promotes bacterial growth Removing the water by a gentle vacuum will decrease the number of times cleaning is needed The BSC chamber should be periodically cleaned by pass ing 100 200 ml of 1N HCI through both the base unit and the top unit being used Immediately after the acid wash 200 ml or more of distilled water should be run through the system DO NOT allow the HCI solution to remain in the chamber DO NOT use the organic solvents including ethanol to clean the chamber as these substances can damage com ponents and seals Publication 5407 001 REV D www harvardapparatus com
8. and thus the fluid level of the chamber Publication 5407 001 REV D www harvardapparatus com 4 Harvard Apparatus Brain Slice Chamber System User s Manual BSC HT Haas Top 4a 4f 4b 4c 4d 4e Figure 4 The BSC HT Haas Top The BSC HT Figure 4 is an interface type chamber which provides rapid fluid exchange and stability for intracellular recordings Slices rest on elastic nylon mesh around which a thin sheet of physiological saline flows by capillary action Slices are oxygenated by a warmed and humidified mixture of 95 02 5 CO2 which flows across the top surface of the slice A flow rate of 0 5 ml min is recommended Step HT 1 To assemble the BSC HT position the top unit so the two clear plastic tubes protrud ing through the top of the base unit Fig 1c and 1d can be connected to the two fit tings on the bottom of the BSC HT Fig 4a After these connections have been made place the top unit onto the base unit Although the top unit is machined to fit into the round opening at the top of the base unit it is recommended that dental wax in addi tion to the two nylon hold down screws be used to secure the top unit Publication 5407 001 REV D www harvardapparatus com 4 Harvard Apparatus Brain Slice Chamber System User s Manual BSC HT Haas Top Step HT 2 An opening has been machined through the side of the top unit Fig 4b for placement of a thermistor probe near the inflow port The thermistor p
9. be negligible Once assembled the chamber is connected to the sources of perfusing solution by the luer fittings located at the bottom of the base unit Flow rates of 0 5 mil min per cham ber can be obtained by keeping the solutions in bottles pressurized to approximately 0 2 kg CM2 by a 95 02 5 CO2 gas mixture Electronically switched valves are rec ommended for solution changes to minimize disruption of solution flow Publication 5407 001 REV D www harvardapparatus com Harvard Apparatus Brain Slice Chamber System User s Manual BSC HT Haas Top Figure 6 Placement of tbe Tbermistor Probe The bubblier port the center elbow on the bottom of the chamber base BSC BU Fig 1a is to be connected to a source of 95 O2 5 CO2 The rate of flow should be adjust ed so the flow across the slice keeps the top surface moist and oxygenated A high gas flow rate which is too high may produce bubbles which are too large and the gas will be inadequately moistened resulting in drying After the gas is bubbled through the water in the base unit it enters the top through the port at the inflow end of the top Fig 4f and passes over the slices The slotted top plate should be adjusted to cover as much of the slice as possible while allowing room to position microelectrodes Proper positioning of this plate is necessary to prevent the slice from drying If necessary holes can be drilled in the slotted cover plate in order to gain better access to t
10. e The rapid exchange of inflowing solutions with each top unit allows for rapid adminstration of drugs and changes in the extracellular ionic environment Publication 5407 001 REV D www harvardapparatus com 4 Harvard Apparatus Brain Slice Chamber System User s Manual BSC BU Base Unit 1f Perfusion Output Line le Perfusion Output Line 1d Left Elbow 1 Right Elbow 1a 1b Banana Center Elbow Connectors Figure 1 Tbe BSC BU Base Unit The BSC BU Base Unit The BSC BU Base Unit Figure 1 is adaptable to both the BSC HT Haas Top and the BSC ZT Zbicz Top The base unit consists of a double walled cylinder enclosing a nichrome wire heating element as well as the tubing which carries the superfusing solution to the top unit Step BU 1 When using the BSC HT the cylinder must be filled with distilled water When using the BCS ZT either distilled water or mineral oil may be used If there is a chance that both the BSC ZT and BSC HT will be used distilled water should be placed in the cylin der as it would be necessary to clean the mineral oil from the gas dispenser tubes before the BSC HT could be used Step BU 2 There are three right angle elbow fittings located on the bottom of the base unit s dou ble walled cylinder The center elbow Fig 1b is used to bubble a 95 O7 5 CO3 mixture through distilled water during operation with the BSC HT it is not used with the BSC ZT The right and left hand elbows are each intended t
11. he slice Also a piece of filter paper should be inserted between the cover plate and the top of the chamber This is to prevent water droplets from accumulating on the inside of the cover plate and dropping onto the slice The electrical ground Ag wire Fig 4c is supplied and should be positioned as near the slice as possible through the insert hole nearest the center of the chamber downstream of the thermistor port Before beginning your experiment this silver wire should be chlorided using the following procedure Chloriding Silver Wire Unused Ag wire should be clean before chloriding This is easily achieved by wiping with alcohol Chloriding the wire is achieved by making it positive relative to a solution containing NaCl 0 9 or KCI G M and passing a current at a rate of 1 mA cm2 of surface area for a minute or until adequately plated a 2 cm length of the 0 25 mm wire used in the holders would require 0 15 mA The color of a well plated wire should be light gray Occasionally reversing the polarity for several seconds while plating tends to yield a more stable electrode Publication 5407 001 REV D www harvardapparatus com Harvard Apparatus Brain Slice Chamber System User s Manual BSC PC Prechamber 7b Figure 7 Tbe BSC PC Precbamber An alternate method of chloriding is to immerse the wire in Clorox until a light gray color is observed typically 15 seconds to several minutes Regardless of the technique used r
12. inse the electrode after plating and store or use A wide mouthed pipette made by cutting a Pasteur pipette can be used to transfer slices from the holding bath to the chamber Slices should be positioned in the longer grooves of the green plastic frame Slices may be transferred to the chamber with solu tion flowing The fluid level in the chamber can be adjusted by placing a piece of tis sue at the edge of the frame to act as a wick The prechamber Figure 7 is designed to maintain 8 12 slices viable for several 6 8 hours prior to transfer to the brain slice chamber 1 Attach one end of a hose to the gas inlet port on the side of the prechamber Fig 7a and the other end of the hose to a source of 95 O2 5 CO2 Do not start the gas flow yet 2 Fill the prechamber to within 1 16 inch of the top with physiological saline maintained at room temperature 3 Using a wide mouth pipette transfer the cut slices to the prechamber by lowering the pipette into the large opening at the top Fig 7b to the proximity of the nylon mesh and release the slice into the mesh 4 Turn on the gas input pressure should not be more than 5 psi Adjust the flow using the valve provided Fig 7c so that gas flows vigorously but not to the extent that the turbulence causes spillage The bubbling gas oxygenates the saline and creates a circular fluid flow which continuously enriches the slices Publication 5407 001 REV D www harvardapparatus com 4
13. itoring and control instruments Most of our products fall into either Category 8 or 9 and are currently exempt from the RoHS Directive We will continue to monitor the application of the RoHS Directive to its products and will comply with any changes as they apply Do Not Dispose Product with Municipal Waste e Special Collection Disposal Required 4 Harvard Apparatus Brain Slice Chamber System User s Manual Table of Contents SUBJECT PAGE NO Warranty and Repair Information 112 1 1 x 2 INtrodUCHON aoi Eb ab Oi 3 BSC BU Base Unb aw o s or ccc 4 5 BSC ZT Chamber Zbicz Top 2 s vssssss1 6 7 BSC HT Chamber Haas Top cccscccssseeeeneees 8 10 BSC PC Prechamber cccccccccssesssssssssssseesseeenes 11 Cleaning Instructions 1111 ssssssssssssnnennnn 12 1 The BSC BU Base Unit ss ssssssssssxxvx 4 2 The TC 202A Temperature Controller 5 3 The BSC ZT Chamber Zbicz Top 111 1 11 7 4 The BSC HT Chamber Haas Top 11 111 8 5 The Brain Slice Chamber System 1 1 9 6 Placement of the Thermistor Probe 10 7 The BSC PC Prechamber 12 11122 un 11 Publication 5407 001 REV D www harvardapparatus com 4 Harvard Apparatus Brain Slice Chamber System User s Manual Warranty and Repair Information Serial Numbers All inquires concerning our product should refer to the serial number of
14. o be connected to the drug s being studied The drug applied to the right hand elbow Fig 1c will flow out of the right hand tube at the top of the cylinder and along the right hand cham bers of the Haas Top the drug applied to the left elbow Fig 1d flows from the left tube at the top of the cylinder and along the left hand chamber of the Haas Top When using the BSC ZT only the right elbow is used care should be taken to plug the cen ter elbow so that fluid within the BSC BU does not leak out Publication 5407 001 REV D www harvardapparatus com 4 Harvard Apparatus Brain Slice Chamber System User s Manual BSC BU Base Unit Figure 2 TC 202A Temperature Controller Step BU 3 The nichrome wire is connected to two banana connectors on the side of the base unit Fig la The base unit may also be connected to a Temperature Controller such as the Harvard Apparatus Inc Model TC 202A Figure 2 which is designed to main tain the perfusate temperature within 0 2 C The resistance of the nichrome wire is approximately 2 ohms should the researcher choose another method of applying cur rent The power source should be capable of delivering approximately 2 5 amps Since the TC 202A is a Bi polar Mono polar Temperature controller and the Base Unit uses a resistive heating element the temperature controller must be set to its Mono polar function The mono polar function can be achieved by setting the switch in the back of the TC
15. robe is inserted through the luer fitting It should be fully inserted so that the thermistor tip is positioned near the center of the groove designed Figure 5 to acc mmiodate it Slices rest The BSC BU with BSCHT BSCZT and BSC PC on elastic nylon mesh panty hose type which must be installed by the investigator along the bottom surface of the green plastic frames which are supplied see Figure 6 To mount the nylon mesh cut a piece of mesh approximately 5 x 11 cm Gently stretch one end of the mesh under the side of the green plastic frame with the milled out channels and push the frame and mesh into the groove of the BSC HT The frame should be above the mesh and positioned on the horizontal portion of the groove The small circles cut in the frame Fig 4d are the prechambers and should be positioned above the inflow ports farthest from the lip of the chamber Fig 4e The remain ing mesh should hang over the lip of the top unit Cut the section of mesh which hangs over the edge to form a point The solution leaving the chamber will drip from this point Alternatively it is possible to use a smaller piece of nylon mesh under the frame and use a second piece of mesh or tissue paper to carry solution over the lip of the chamber The BSC HT is designed so that the effects of two drugs can be studied simultaneous ly one drug flowing down the left side of the chamber and the second down the right Intermixing has been found to
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