CryoJane User Manual
Contents
1. CryoJane User Manual Customer Service 1 800 237 2772 In continental US only Customer Service 1 201 343 1313 International Instrumedics grants the purchaser of the CryoJane system the rights to use the patented Cryo Jane CFSA materials and process Patent 4 545 831 CRYOJANE TAPE TRANSFER STEP BY STEP INSTALLATION INSTRUCTIONS The following items are required for installation Absolute Alcohol 1600 Watt Hair Dryer Gauze Gloves A EZ T D m ml he LE ba 01 Remove release paper 02 Place ECU on top left 03 Using absolute alcohol and gauze from ECU corner of cryostat clean chamber wall 04 Heat left wall for Mech amp 05 Place Oil Bath towards the 06 Route Oil Bath amp Mech Cables Oil Bath placement rear of the left chamber wall underneath Mech 07 Place Mech in Chamber 08 Route cables in door bar 09 Place door bar with cables Place Mech low in chamber onto front chamber ledge near the front wall If required attach Door Bar Support Strip Placement near the freeze underneath door bar bar is ideal in most cryostats R SUPPLY a TRUMEDICS 10 Adhere Cable holders 11 ECU Rear with Cable amp 12 Connect Mech Oil Bath Cables to along cryostat top outer wall Power Connectors ECU amp Power cord to the ECU and wall outlet Turn the ECU on pa ic E an 13 Mech Cable 14 External Installation 15 Internal Installation Complete connected to ECU Complete Cryo Vac Sh2. THE CRYOSTAT CHAMBER TO AVOID WARM AIR CONTACTING AND MELTING THE FROZEN SECTION 28 The initial freezing was poor slow causing large ice crystals to grow The section was taken deep in the tissue block where the freezing is the poorest Snap freeze tissue at 60 C or colder Using Instrumedics Power Jane or Stand Alone Gentle Jane or Dewar Jane snap freezers with liquid nitrogen produces the best results Snap freeze the thinnest practical specimen The sections uppermost in the block will have the minimum ice crystal artifact Use Instrumedics Aqueous Fixative NOTE Excellent results with freeze substitution followed by anhydrous fixation will occur only if the ice crystal size is minimal 29 rz p Fei os Teita 7 T A at pee i E Til jfi 4 mye ts e a 4 a 1 FIGURE 28 Oil Bath Accessory Kit Components Oil Bath Oil Oil Brush Oil Bath Power Cable not shown 30 Oil Bath Accessory Kit Components Oil Bath Accessory Kit Operating Procedure Background Virtually all embedding media are water based When a frozen tissue specimen embedded in such media is stored over time the tissue and the medium will dehydrate and degrade The Oil Bath Accessory Kit enables the user to coat the block face with the protective Oil that is maintained at 8 C in a bath inside the cryostat chamber Coating the surface will not melt the tissue in the block The Oil h
3. freezing In such cases the aqueous fixative protocol should be followed An anhydrous fixative contains a maximum of 30 water 1 AN DMEF Glut Dimethylformamide DMEF 35 ml Acetonitrile or AN 35 ml 25 glutaraldehyde 10 ml 50 Glut 5 ml Add H O to make up to 100 ml Wait at least 1 our before use 2 Formal Ethanol Ethanol 100 alc 86 ml Formaldehyde 14 ml Bring the fixative into the cryostat and transfer the freeze substituted slide directly from the acetone bath into the fixative Continue fixation at room temperature for 15 to 30 seconds Transfer is made within the cryostat to prevent condensation of water on the cold slide prior to fixation 21 3 For very sensitive antigens DMF Glut Dimethylformamide 70 ml 25 Glutaraldehyde 1 ml Add H O to make up to 100 ml Transfer the freeze substituted slide directly from acetone to this fixative at 4 C for 30 to 60 seconds This transfer must be made within the cryostat Following fixation the slides are rinsed in water prior to staining REFER TO REAGENT MSDS FOR SAFE HANDLING CryoJane Maintenance ECU NOTHING SHOULD BE PLACED OR STORED ON TOP OF THE ECU Any spills which enter the ECU may severely damage the electronic components To clean the ECU wipe the surfaces with either a dry rag or if necessary an alcohol wipe ACETONE OR OTHER SOLVENTS WILL DAMAGE THE PLASTIC SURFACES Fuses can be replaced by unscrewing the two small fuse holders
4. of the block by moving the Roller up and down one to two times and then left to right one to two times Some pressure is required for good adhesion When finished place the Hand Roller back to its original position inside the cryostat F igure 11 Laminating the Tape to the block face 14 8 Turn the flywheel slowly and evenly to cut a section If a sharp knife is used the section captured on the Tape will be flat intact uncompressed and as thin as two microns No need for a brush or an anti roll device F igure 12 Cutting the section 9 Pick up the Tape at either tab and transfer it to the Slide on the Blue Pad making sure to keep it down inside the cryostat to avoid melting the section 10 Position the Tape section side down on the Adhesive Coated Slide For multiple sections refer to the Steps for Multiple Sections 15 Figure 14 Positioning the Tape on the Slide 11 Use the cold Hand Roller to laminate the Tape to the Slide Apply even pressure on the Roller and roll it over the section under the Tape two or three times Figure 15 Laminating the section to the Slide 12 Holding the frosted end of the Slide remove it from the Blue Pad and transfer it into the Flash Tray Gently push the Slide in as far as it will go against the stop To check lift the Lid Close before actuating the flash Figure 16 Placing the slide into the Flash Tray 13 Push and release the Black Knob
5. seat the Slide on the Flash Tray The Tape tabs will overhang the Flash Tray 18 Figure 20 Technique for removing Tape from Slide Figure 21 Processing multiple sections 19 Close the Lid and push and release the Black Knob to actuate the UV flash Then lift the Lid remove the Slide and carefully peel away each strip of Tape as described in Steps 15 and 16 Figure 22 Curing multiple sections Summary of the Instrumedics Frozen Sectioning Process Controlled drop Gentle Jane Device Om Block FIGURE 23 To minimize ice crystal size tissue is snap frozen with a chilled heat extractor Block Tt ccm Tape So pirataie Knife FIGURE 24 A cold adhesive tape is used to support the section as it is being cut he Adhesive Coated Slide Adhesive Tape FIGURE 25 The still frozen section Snap freezing and embedding A blockholder is placed on the Gentle Jane snap freezing device CryoGel or other embedding media is dispensed onto the blockholder and a tissue specimen is positioned on top The chilled heat extractor is placed in its holder and released When the heat extractor contacts the specimen the tissue and embedding medium is snap frozen into a flat block The plane of the flat block face minimizes trimming When specimen shape or orientation are essential the tissue is snap frozen using the Instrumedics CryoGel Rubber Mold method Cutti
6. anual CryoJane ECU Figure 2 CryoJane ECU back Figure 3 CryoJane ECU front and right side 1 On Off Switch Pad LED Power LED or Bath LED if optional Oil Bath Accessory Kit is installed Flash Pad Connector Optional Oil Bath Connector Fuses A es A a a p AC Power Cord Connector CryoJane Mech Components a agi d aF p io FIGURE 4 CryoJane Mech Components 1 Blue Pad Stainless Steel Spring Clip Black Knob Flash Tray Lid Sa e eR 10 CryoJane Operating Procedure Background In conventional sectioning the operator usually guides the cut section over the knife using a brush or an anti roll device Even with a skillful steady hand this step can be difficult especially if a 2 to 4 micron thick section is desired To mount the section a room temperature slide is touched to it When contact is made between the warm slide and the frozen section instantaneous melting of the section occurs converting the ice crystals back into water Surface tension causes the melted section to adhere to the slide When the ice melts water fills the spaces formerly occupied by the ice crystals rehydrating the nucleoplasm and cytoplasm causing erythrocytes to lyse and soluble substances to be displaced Flow of the melted solutions can distort and displace fine structure Often the melted section is then dried during which step surface tension forces further distort displace and collapse tissue structure Patho
7. as a freezing temperature of 10 C and will instantly freeze on the block face The frozen Oil coating prevents tissue and medium dehydration for up to one year if stored in a 70 C freezer The Oil can also be used to repair a damaged block A deformation or hole in the block face may be filled with the Oil which will freeze The frozen Oil has the property of an embedding medium and can then be sectioned similarly to other media Preparation e The cryostat temperature should be 25 C to 30 C e Make sure there is Oil in the Bath If Oil is added to the Bath wait 1 to 2 hours for the Oil to reach its set temperature of 8 C e The Oil inside the Oil Bath should be in a liquid state not frozen e The Oil Brush should be inside the Oil Bath e The right LED on the ECU labeled Oil Bath should be green 30 Use A tissue block can be stored in a freezer at 70 C for up to year once thoroughly coated with the protective Oil THE BLOCK MUST NOT BE PERMITTED TO THAW AND REFREEZE DURING STORAGE THIS WILL DEGRADE THE QUALITY OF THE TISSUE IN THE BLOCK Storing a Tissue Block To store short term in the cryostat or long term in a freezer a frozen tissue block follow the procedure outlined below For Short Term Storage in the Cryostat With the block in the cryostat use the Oil Brush to coat the block face with the Oil from the Oil Bath For Long Term Storage in a 70 C Freezer With the block in the cryostat use th
8. ck for looseness and tightened tighten all screws and clamps as needed The captured section on the Tape is not intact Cause Solution If using a disposable blade shift to a different The knife is dull portion of the blade s edge or replace the blade If using a stainless steel or tungsten carbide knife sharpen the knife There is dirt on the Tape Use a fresh clean and cold Tape Bubbles form while laminating the Tape to the Apply the Tape like wallpaper to the block block face face while laminating with the cold Roller If bubbles are still visible reapply the Tape There is vibration in the microtome thick thin Check the knife and block for looseness and chatter etc tighten all screws and clamps as needed The section on the Tape does not transfer properly to the Slide Cause Solution The mylar film on the Slide has not been Remove the mylar film removed The adhesive layer on the Slide is not facing Position the Slide on the Blue Pad so the up adhesive layer faces up See the Mech and ECU Troubleshooting The Slide is too cold or too warm Guide for the left LED on the ECU labeled Pad always displaying either a solid red or flashing red light 26 The Tape is not being removed properly The knife is dull A bubble may have formed when the Tape was laminated to the Slide Remove the Tape slowly and carefully peeling it diagonally and downward to minimize the tension on the sectio
9. d Cause The ECU is not turned on The 2 inch diameter Flash Pad Power Cable is not connected properly One or both fuses in the back of the ECU are blown The Flash triggering mechanism is jammed 24 Solution Make sure the ECU is properly connected to an electrical source Make sure the On Off Switch on the ECU is in the On position pushed in If the LED s on the front of the ECU are on that indicates that the ECU is on Make sure the Flash Pad Power Cable is properly connected to the ECU Check the fuses on the back of the ECU and replace as needed NOTE If flash unit is triggered more than 3 times in one minute one or both fuses on the back of the ECU will blow out and will need to be replaced This is a protective feature designed to prevent overheating Use only IAMP SLO BLO Ceramic Core Wound fuses available from Instrumedics Check for ice build up around the Black Knob on the Mech and remove if found If this problem cannot be corrected call Instrumedics 800 customer service number The flash unit is malfunctioning The ECU is malfunctioning and arrange for servicing of the triggering mechanism 1 800 237 2772 Call Instrumedics 800 customer service number and arrange for servicing of the flash unit 1 800 237 2772 DO NOT OPEN THE FLASH UNIT Call Instrumedics 800 customer service number and arrange for servicing of the ECU 1 800 237 2772 DO NOT OPEN THE ECU CryoJane Process T
10. e Oil Brush to coat the entire block with the Oil from the Oil Bath Apply generous amounts of Oil Figure 29 Coating the block with Oil Sectioning a Stored Tissue Block From Short Term Storage Trim away the frozen layer of Oil to expose the tissue specimen and proceed as usual 31 Oil Bath Accessory Kit Troubleshooting Guide The Oil inside the Oil Bath is frozen Cause The ECU is not turned on The Oil Bath Power Cord is not connected properly One or both fuses on the back of the ECU are blown The cryostat temperature is too cold The ECU is malfunctioning Solution Make sure the ECU 1s properly connected to an electrical source Make sure the On Off Switch on the ECU is in the On position pushed in If the LED s on the front of the ECU are on that indicates that the ECU is on Make sure the Oil Bath Power Cord is properly connected to the ECU and the Oil Bath Check the fuses on the back of the ECU and replace as needed Use only IAMP SLO BLO Ceramic Core Wound fuses available from Instrumedics Raise the cryostat temperature Call Instrumedics 800 customer service number and arrange for servicing of the ECU 1 800 237 2772 DO NOT OPEN THE ECU The Oil Bath LED always displays a solid red light Cause The Oil Bath Power Cord is not connected properly The temperature of the Oil inside the Oil Bath is too cold The ECU is malfunctioning 32 Solution Make sure
11. ebris was captured between the adhesive Tape and the block face or between the Tape and the coated Slide A hole in the block face may be due to groups of cells being plucked out during or prior to sectioning Debris was caught between the Tape and block or between the Tape and the coated Slide Solution To avoid bubbles apply Tape like wallpaper If bubbles are visible under the Tape on the block face reapply the Tape Use clean Tape This may be a defect in the tissue block Check the knife edge for dullness and correct Always remove all trimming debris from the block face and both the front and rear surfaces of the knife s cutting edge before sectioning The section did not transfer completely See the CryoJane Process Troubleshooting Guide There are holes in the nuclei and or cytoplasm of the section Cause Section may have melted and refrozen causing large ice crystals to grow This is the most common problem Solution Transfer the frozen block from the freezing device rapidly to the cryostat to avoid thawing the tissue Avoid finger contact with the central portion of the Tape and or Slide Do not touch the block face with your fingers thumb Do not breath directly on the section Keep Tapes and Slides inside the cryostat The Blue Pad temperature may be too warm See the Mech and ECU Troubleshooting Guide ALL STEPS OF THE Cryo Jane PROCESS MUST BE CARRIED OUT DEEP INSIDE
12. en E E ander ethene E deuiee 22 Troubleshooting Guide 0000 00 oe ce ee ee ee 23 Troubleshooting the Mech and ECU ccc c cece cece eee nee e eee e ee ees 23 CryoJane Process Troubleshooting ccc cece cece eee e eee e eee eeeeeenees 25 Troubleshooting Finished Slides ccc ccc cccc eee e eee e cece eeeeeneeeeeees 2 Oil Bath Accessories Kit Components 00 29 Oil Bath Accessory Kit Operating Procedure 30 BACKOnOUNC o teauMemsnutateaaunedundakneu te edeutatcuuun mbasdatces E 30 PPE al AION snatches toy Sola caresses Carpe aba Soke N Sakae kere 30 NISC E IE EE E Neto citrate aGhvona tated ow A tates eben eels 31 SOM aT issue BIOCK aaier uneei an aE 31 Sectioning a Stored Tissue Block cc cece cece cent eee eee ees 31 Oil Bath Accessory Kit Troubleshooting Guide 32 Instrumedics Frozen Section Process Overview Freezing Process Conventional Freezing vs Snap Freezing In the conventional freezing method the tissue is typically frozen at about 30 C During this relatively slow freezing process the ice crystals that form are usually large compared to cellular dimensions These crystals can cause considerable displacement and structural damage to the tissue The Gentle Jane device is designed to snap freeze the tissue in approximately 8 10 seconds The freezing agent can be liquid nitrogen LN chilled isopentane dry
13. from the knife Set the micrometer for the desired section thickness and cut two or three sections to be sure a full section will be cut Take a pre cooled pouched Adhesive Coated Slide and while holding it inside the cryostat chamber remove the glass slide from the pouch oo oY Figure 6 Adhesive coated slide in pouch 12 3 Holding the Slide at the frosted end rapidly peel off the protective mylar film which covers the adhesive and discard it Figure 7 Peeling mylar from adhesive coated slide 4 Place the Coated Slide adhesive side up on the Blue Pad of the Mech by slipping the frosted end of the slide under the stainless steel Spring Clip 5 Inside the cryostat pick up a pink Tape and peel away the protective film covering the adhesive window Discard the protective film Pa Figure 9 Exposing the Tape window 13 6 Hold the Tape up by the narrow tab with the adhesive side towards the block face Position the Tape in front of the trimmed block so that the bottom edge of the exposed adhesive is aligned with the bottom edge of the block Place the Tape on the block face making sure that the bottom wider tab of the Tape overhangs the knife NO PORTION OF THE ADHESIVE WINDOW SHOULD OVERHANG THE BLOCK FACE TO AVOID MELTING THE SURFACE OF THE TISSUE DO NOT TOUCH THE PORTION OF THE TAPE THAT COVERS THE BLOCK FACE 7 Using the cold Hand Roller laminate the Tape to the face
14. ice or a thermoelectric cooler Peltier device The temperature at which the tissue is frozen should be 60 C or colder NOTE Gentle Jane Snap Freezing methods are described in a separate Snap Freezing User Manual CryoJane Process The CryoJane process is built around three special methodologies 1 The capture of an undistorted thin snap frozen section on a special cold adhesive tape 2 The lamination of the captured section onto a cold glass microscope slide coated with an ultraviolet light curable pressure sensitive adhesive 3 The curing of the adhesive on the slide with an 8 millisecond ultraviolet flash and the subsequent removal of the adhesive tape leaving a still frozen section firmly adhered to the microscope slide The CryoJane system is designed to permit the facile application of these methodologies and can be adapted to virtually all model cryostats CryoJane System Components FIGURE I CryoJane System Components l Mechanism Mech See CRYOJANE MECH COMPONENTS for details Electronics Control Unit ECU See CRYOJANE ECU for details 2inch diameter Mech Cable connected to the Mech Door Channel 3 Shelf Unit Hand Roller Adhesive Coated Slides Tape Windows et Ses e SS a Knife Facet Wipers 10 AC Power Cord not shown OPTIONAL EQUIPMENT 11 Protective Oil Accessories Kit see Oil Bath Accessories Kit sections in this manual 12 Cryo Vac Away System see Cryo Vac Away User M
15. in the rear of the ECU Fuses should be 1 AMP SLO BLO and ceramic core wound available from Instrumedics DO NOT OPEN THE ECU Only personnel authorized by Instrumedics should service the ECU Unauthorized opening of the ECU will void all warranties Unauthorized persons will assume all risks and responsibilities Mech All metal surfaces may be wiped with alcohol or chlorine solution The Flash Tray should periodically be cleaned with alcohol to remove any adhesive build up Care must be taken to avoid damaging the glass filter in the Flash Tray 22 e Liquid spills on the Mech should be wiped immediately Electronic components in the Flash Lamp may short circuit if exposed to liquids e DO NOT IMMERSE THE MECH IN ANY SOLUTIONS e To remove the Mech from the wall for decontamination or servicing simply unscrew the two screws shown below and then remove the Mech from the cryostat The Mech s mounting plate will remain attached to the wall of the cryostat Tip To avoid losing the two screws screw them into the holes in the mounting plate remaining on the wall To reinstall the Mech attach it to the mounting plate with the two screws lt a 2 a A Troubleshooting the Mech and ECU The LEDs lights on the ECU are not on Cause Solution Make sure the ECU is properly connected to an electrical source Make sure the On Off The ECU is not turned on Switch on the ECU is in the On position pushed in If the LED
16. logists and clinicians have been trained to recognize normal and abnormal structure against this background of degraded fine structure In contrast the CryoJane process is designed specifically to avoid melting and air drying and its associated artifacts It uses a cold adhesive tape to support and capture the section during the cutting step The unthawed section on the tape is then transferred to a special cold adhesive coated slide The section is not melted in the mounting step The adhesive coating on the slide is then polymerized and the tape is peeled away leaving the section permanently anchored to the slide while still frozen Preparation e Aday s supply of Adhesive Coated Slides and Tape Windows should be in the cryostat chilled and ready for use e The cryostat temperature should be 25 C to 30 C depending on the cryostat model e The Pad light left LED on the ECU should be green 11 The right LED should be red if it is labeled Power and green if it is labeled Oil Bath Use Once a tissue specimen has been frozen and mounted in the microtome chuck follow the procedure outlined below Steps for a Single Section l If the Cryo Vac Away is installed position the collection nozzle at the knife edge and step on the foot switch to automatically remove debris during the trimming sequence If the Cryo Vac Away is not installed trim the block to the desired depth to obtain a full block face and remove the debris
17. n If using a disposable blade shift to a different portion of the blade s edge or replace the blade If using a stainless steel or tungsten knife sharpen the knife Apply the Tape like wallpaper to the Slide while laminating with the cold Roller Troubleshooting Finished Slides The polymerized adhesive picks up excessive red stain after H amp E Cause Eosin may be too acidic or too alcoholic This may cause background staining Solution Stain a shorter time or use an aqueous eosin Y at pH 5 0 5 2 There are chatter marks venetian blind effect parallel to knife edge in the section Cause There is looseness in the system The knife is dull Stop start motion during cutting There are tears in the section Solution Check tightness of chuck Clamp knife holder and or blade holder very tightly If using a disposable blade shift to a different portion of the blade s edge or replace the blade If using a stainless steel or tungsten knife sharpen the knife Cut slowly without stopping If motor drive is used set cutting speed slower Cause Solution 2 The knife has a nick or defect on the cutting edge If using a disposable blade shift to a different portion of the blade s edge or replace the blade If using a stainless steel or tungsten knife sharpen the knife Small clusters of cells usually circular are missing in the section Cause Bubbles or d
18. ng After the block is trimmed a cold adhesive tape is adhered to the block face The tape supports and captures the section as it is being cut eliminating the need for a brush or anti roll device Transfer to Slide A cold adhesive coated slide is placed on a temperature controlled pad The adhesive tape is placed section side down on the adhesive coated slide and is laminated to the adhesive layer using a cold roller 19 adhered to the tape is rolled onto the cold adhesive coated slide U V light FIGURE 26 An ultraviolet flash converts the adhesive coating into a hard solvent resistant plastic ae FIGURE 27 The tape is removed Several options are available Curing the Adhesive Coating A flash of ultraviolet light passes through the slide to polymerize the adhesive layer on the slide into a hard solvent resistant plastic tightly anchoring the section to the slide Removal of Tape The tape is peeled away leaving the still frozen section tightly bonded to the plastic layer The slide can then be a fixed in the Instrumedics Aqueous Fixative b freeze dried or freeze substituted in the cryostat before anhydrous fixation c melted or air dried and fixed with the fixative of your choice Special Fixation Protocols Aqueous Fixative THIS FIXATION SHOULD BE USED FOR ALL HISTOLOGICAL STAINING OF CRYOJANE PREPARED SECTIONS This fixative melts and fixes the tissue simultaneou
19. own Cryo Vac Shown The standard CryoJane Mech installation is shown in Fig 07 The Hacker Bright OTF and Microm HM 560 cryostats require an S Plate Mech and separate mounting plate for installation S Plate Mech for Hacker OTF and Microm HM 560 Cryostats Mounting Plate locks onto S Plate Mech Slide Nest Entrance The entire assembly is adhered to the left cryostat wall as shown in Fig 07 Note The S Plate Mech must have enough clearance from the front wall of the cryostat for slides to enter the Slide Nest Table of Contents Instrumedics Frozen Section Process Overview 7 Pr C7 PROC CS 6 acre ete ged tutnch cnr a E 7 Coane OCS Siurana ra E E sus eeeeaanaean bina cesaee 7 CryoJane System Components 0 000 ccccceeceee eee 8 CiyoJane ECU oeren NN ne ren 9 CryoJane Mech Components 0 0cccceeeceeeee 10 CryoJane Operating Procedure 0 e 11 Bock CrOUNG eeen E E E E san 11 Pie o O E AEE A A EE I TEE A ET A EE TE 11 D PE EEEE EE ET IA EEE ETEA ESEE T 12 Steps rora S mele SCCHOM sericea a E T 12 Steps for Multiple Sections aenea EAA a 18 Summary of the Instrumedics Frozen Sectioning Process 19 Special Fixation 0 aaaea aae ee ee ee aes oes oes oti wee uori ne 20 Adg eous FixaItiOm n araa ea aa a tad ended Geedauk ben 20 ANDY OUS FIANO eies N O E A 21 CryoJane Maintenance 00000000 00 cece cece cee tee eee eeeee 22 POU sere b
20. roubleshooting The CryoJane process will not produce the desired results if the cryostat and or the microtome are malfunctioning Both need to be serviced periodically Check with the manufacturer dealer The Tape does not adhere to the block face Cause The protective film covering the adhesive is not removed The block is too cold or too warm There is moisture on the adhesive layer Solution Peel away the protective film covering the adhesive window Check the temperature of the cryostat and adjust if necessary Chamber temperature should generally be set between 25 C to 27 C depending on the particular cryostat model Use a fresh Tape The Tape does not capture the section Cause The microtome is not advancing Solution Check the microtome and adjust The microtome is not advancing at the desired Check the microtome and adjust Cut two or thickness The knife angle is too small or too large three sections before applying the Tape Adjust the knife angle between 3 and 5 25 degrees The Tape is defective Use another Tape The Tape is cut during sectioning Cause Solution The block face is not flat Align and trim the block to obtain a flat face The adhesive on the Tape overhangs the block Position the Tape on the block so that no and snags on the knife adhesive is exposed to the knife when cutting the section The knife and or the block are not be properly Check the knife and blo
21. s are on that indicates that the ECU has power Check the fuses on the back of the ECU and One or both fuses in the back of the ECU are replace as needed Use only IAMP SLO blown BLO Ceramic Core Wound fuses available from Instrumedics Call Instrumedics 800 customer service The ECU is malfunctioning number and arrange for servicing of the ECU 1 800 237 2772 DO NOT OPEN THE ECU The left LED labeled Pad always displays a solid red light Cause Solution The 1 2 inch diameter Flash Pad Power Cable is Make sure the Flash Pad Power Cable is not connected properly properly connected to the ECU and to the Blue 23 The temperature of the Blue Pad is too cold The ECU is malfunctioning Pad connector underneath the Mech Raise the cryostat temperature Call Instrumedics 800 customer service number and arrange for servicing of the ECU 1 800 237 2772 DO NOT OPEN THE ECU The left LED labeled Pad always displays a flashing red light Cause The temperature of the Blue Pad is too warm The ECU is malfunctioning Solution Lower the cryostat temperature Instrumedics 800 customer Call number and arrange for servicing of the ECU 1 800 237 2772 DO NOT OPEN THE ECU Service ONLY IF THE OIL BATH OPTION IS INSTALLED The right LED labeled Oil Bath is always red Refer to the Oil Bath Accessory Kit Troubleshooting Guide in this manual The Flash does not go on when triggere
22. sly direct controlled melting Ice crystal artifacts holes are masked Nuclear morphology is usually excellent Some cell components are lost and most of the red blood cells are lysed 1 PREPARATION OF AQUEOUS BUFFER Dissolve Buffer Salt Mix in 180 ml of water Buffer Salt Mix is available from Instrumedics Cat AB Store in refrigerator Usable for at least 6 months 2 PREPARATION OF AQUEOUS FIXATIVE Add 20 ml of 25 glutaraldehyde to 30 ml of Aqueous Buffer Mix well Use at room temperature Discard after 1 week If using 50 glut add 10 ml glut and 10 ml water CAUTION 3 PROCEDURE Contains Cacodylic Acid Avoid contact with fingers or skin Do not breathe dust from vial 20 a After the adhesive coating on slide has been polymerized carefully peel off the pink Tape b To avoid uncontrolled melting and or drying of the section immediately bring the room temperature aqueous fixative into the cryostat and immerse the slide into the fixative Dip 2 3 times c Continue to fix for 15 to 30 seconds at room temperature d Rinse the slide in water and proceed with a chosen staining protocol Anhydrous Fixation Protocol To be used after freeze substitution in a cold solvent such as acetone at 30 C or colder Anhydrous fixation preserves most cellular components and is highly recommended for routine stains Note Not recommended when ice crystal size is large and widespread poor
23. the Oil Bath Power Cord is properly connected to the ECU and the Oil Bath Raise the cryostat temperature Call Instrumedics 800 customer service number and arrange for servicing of the ECU 1 800 237 2772 DO NOT OPEN THE ECU The Oil Bath LED always displays a flashing red light Cause Solution The temperature of the Oil inside the Oil Lower the cryostat temperature bath 1s too warm Call Instrumedics 800 customer service The ECU is malfunctioning number and arrange for servicing of the ECU 1 800 237 2772 DO NOT OPEN THE ECU 33
24. to actuate the UV flash The UV cures the adhesive on the Slide into a polymer anchoring the frozen section to the Slide Figure 17 Curing the adhesive on the Slide 16 14 Holding the frosted end immediately remove the cured Slide from the Flash Tray DO NOT TAKE THE SLIDE Boii ap OUT OF THE CRYOSTAT AT THIS ee nae Be eae TIME AS THAT WILL CAUSE THE rp Va pee ae SECTION TO MELT 15 Keeping the Slide deep inside the cryostat chamber carefully remove the pink Tape to expose the frozen section on the Slide Figure 19 Removing Tape from cured Slide 16 Peel off the Tape diagonally and downward to minimize the tension on the section and to insure complete transfer Do not touch the section with your fingers Once removed discard the Tape The slide can now be 17 a fixed in the Instrumedics Aqueous Fixative b freeze dried or freeze substituted in the cryostat before anhydrous fixation Note The section should be immersed in the fixative inside the cryostat Fixation should then continue at room temperature c melted or air dried and fixed with the fixative of choice for your protocol Steps for Multiple Sections 17 18 Multiple sections can be positioned on one Slide by placing the Tapes perpendicular to the Slide For small tissue specimens the Tape can be cut into narrower strips Follow Steps 5 9 To cure the adhesive on the slide lift the Lid of the Mech and
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