Brain Research Rullrrin.
Vol.
25, pp. 437+Kl.
C
Pergamon Press plc,
1990.
036L9230190
Printed in the U.S.A
$3.00
t .OO
BRIEF COMMUNICATION
Cryostat Sectioning of Large Brains Made Easy IVAN DIVAC,’ EGIL GUDBRANDSEN, KURT HELMER. JESPER MOGENSEN AND PHILIP LARSEN
Institute of Neurophysiology, University of Copenhagen School of Medicine. 2200 Copenhagen, Denmark Received
1 May 1990
DIVAC. I., E. GUDBRANDSEN, K. HELMER, J. MOGENSEN AND P. LARSEN. Crwsrat secrioning oflarge hrnins made ensy. BRAIN RES BULL 25(3) 437440, 1990.-The present paper describes: I) an easy and reliable method to mount cryostat sections from large brains and 2) modifications of a cryostat to make sectioning faster and simpler. Cryostat
CRYOSTAT methods and
improvements
sectioning obligatory
Method of mounting
is preferable
for
some
Cryostat
sectioning
modifications
and curvature are to be adjusted to the microtome. Thus obtained sections (we used only fixed tissue) were mounted with help of a frame and a block (Fig. 2). The frame consists of a firm aluminum plate which has an L-shaped holder glued to each side of the plate and is made of pertinax which is a good thermal insulator. Thus, the warmth of the hand is not transmitted to the aluminum plate. The block is made of stainless steel. has a holder on the upper surface and a flat, teflon-coated lower surface. A thin layer of teflon (PTFE) was sprayed on the steel (thickness about 0.02 mm) and was hardened by baking at 390°C. The section to be mounted is placed on an alum-gelatine coated object glass (or coverslip, for the 2-deoxyglucose method) and together with it put on the aluminum plate of the frame. This sandwich, with the section above the glass, is topped by the block. then taken out of the cryostat and laid on a hot plate heated to about 60°C. At the same moment. the block is pressed against the section causing its adherence to both glass (below) and teflon (above). Delayed or insufficient press leaves bubbles under the section (Fig. 3A and B). Immediately afterwards. the glass with the section is separated from the block by pulling an edge of the glass away. Sliding movements between the teflon surface and the section must be avoided. Precise timing of this procedure depends on the thickness of the section and the glass as well as on the temperature of the cryostate and the hot plate. Short practice suffices to obtain flat sections with very few or no air bubbles (Fig. 3C, D). Two counters and an electronic “window” solved the remaining problems. The sections to be rejected are counted by a stepdown counter (Predetermining counter S-4330 by Coulboum
histochemical
for work with diffusible substances. During our work with a Bright (Hacker in USA) cryostat (the least expensive among those able to cut blocks of the size of the monkey brain) we encountered several problems: First, sections from large brains are notoriously difficult to mount. Considerable patience and dexterity are needed in order to avoid wrinkles or trapped air bubbles. Hand’s (I) solution was to divide large brains into blocks to be cut separately. Secondly, in order to keep constant the distance between saved sections, the technician had to count the sections to be discarded. Any distraction may result in varying distance between mounted sections. Thirdly, the block-advancing mechanism in Bright microtome must be reset after a number of sections is cut. If this count is not precise one must either reset the microtome more often than necessary or risk a time-consuming rewinding of a loose screw. Fourthly, we were wasting time while waiting for a small block to descend slowly along the whole path. Finally, we found that the antiroll plate and the knife-extension plate provided by the manufacturer were inconvenient. Several modifications of the cryostat (model 1980) and a new method of mounting the sections eliminated the problems described above. Among the cryostat modifications, the antiroll plate was enlarged to cover almost the entire space between the knifeholders. The new plate was made of perspex and had the same dimensions, except its breadth, as the original one. The angle between the antiroll plate and the knife was reduced in order to prevent bending the sections. The knife-extension was shaped as a ski-jump ramp in order to slide large sections onto the glass (Fig. 1). The shape of the ramp can be seen in Fig. 1. The dimensions ‘Requests for reprints should be addressed Denmark.
Cryostat
to Ivan Divac, Institute of Neurophysiology.
437
Panum Institute, Blegdamsvej
3C. DK-2200 Copenhagen
N.,
,OBJECT
HOLDER
I
SKI-JUMP
BLOCK
MAL
PLATFORM
1NSULAfiON
FTG. 2. The frame and block used for mounting large sections.
FIG. 1. Modifications of the aatiroll plate and the knife-extension.
Ins~m~nts, Box 2551, Lehigh Valley, PA 18001) which is set at the number of sections to be rejected. After the p~de~~~ number of strokes the serial cutting stops automatically and the section to be mounted is cut and saved. The counter is reset, and another series of sections to be rejected is cut. A cumulative counter counts all strokes and can be used to reset the block-advancing mechanism before the screw falls off. Finally, we provided an electronic “window” which could be
set according to the size and position of the block. The purpose of the window is to have the block move at the highest speed at al# times except while the tissue is being cut. In this way much time is saved, particularly when one must cut all sections at a very low and even speed, as is required by the 2-deoxyglucose method. Although more advanced cryostats incorporate some of the functions we describe above, the diagram of the electronic circuit is given (Fig. 4) for those laboratories which have more manpower than means to buy expensive equipment.
The research which led to the present& described method was supported by the Danish Medical Research Council. We thank Kirsten Lindberg for the drawings, Ulla Mogensen for cutting tbe brains and suggesting improvements, F. Riis for help with photography, Margit L@vgreenfor typing and R. Gunilla E. &erg for comments on the manuscript.
REFERENCE I.Hand, P. J. The 2-deoxyghzcose metbrxi. In: Heimer, L.; RoBa&, M. E., eds. Neuroanatomical tract-tracing methods. New York: Plenum; 198151 l-538.
CRYOSTAT
SECTIONING
OF LARGE
BRAINS
439
FIG. 3. Autoradiograms of monkey sections mounted by the presently described method. A and B illustrate consequence of late or insufficient press of the block on the section at the mc~ment of placing on the hot plate. C and D present successfull); mounted sections. The ma~nifieations of different sections are not equal.
f
+
f
t
e
+ FIG. 4. Diagram of the speed electronic circuitry subserving the window and the counters,
Vol.
25, pp. 437+Kl.
C
Pergamon Press plc,
1990.
036L9230190
Printed in the U.S.A
$3.00
t .OO
BRIEF COMMUNICATION
Cryostat Sectioning of Large Brains Made Easy IVAN DIVAC,’ EGIL GUDBRANDSEN, KURT HELMER. JESPER MOGENSEN AND PHILIP LARSEN
Institute of Neurophysiology, University of Copenhagen School of Medicine. 2200 Copenhagen, Denmark Received
1 May 1990
DIVAC. I., E. GUDBRANDSEN, K. HELMER, J. MOGENSEN AND P. LARSEN. Crwsrat secrioning oflarge hrnins made ensy. BRAIN RES BULL 25(3) 437440, 1990.-The present paper describes: I) an easy and reliable method to mount cryostat sections from large brains and 2) modifications of a cryostat to make sectioning faster and simpler. Cryostat
CRYOSTAT methods and
improvements
sectioning obligatory
Method of mounting
is preferable
for
some
Cryostat
sectioning
modifications
and curvature are to be adjusted to the microtome. Thus obtained sections (we used only fixed tissue) were mounted with help of a frame and a block (Fig. 2). The frame consists of a firm aluminum plate which has an L-shaped holder glued to each side of the plate and is made of pertinax which is a good thermal insulator. Thus, the warmth of the hand is not transmitted to the aluminum plate. The block is made of stainless steel. has a holder on the upper surface and a flat, teflon-coated lower surface. A thin layer of teflon (PTFE) was sprayed on the steel (thickness about 0.02 mm) and was hardened by baking at 390°C. The section to be mounted is placed on an alum-gelatine coated object glass (or coverslip, for the 2-deoxyglucose method) and together with it put on the aluminum plate of the frame. This sandwich, with the section above the glass, is topped by the block. then taken out of the cryostat and laid on a hot plate heated to about 60°C. At the same moment. the block is pressed against the section causing its adherence to both glass (below) and teflon (above). Delayed or insufficient press leaves bubbles under the section (Fig. 3A and B). Immediately afterwards. the glass with the section is separated from the block by pulling an edge of the glass away. Sliding movements between the teflon surface and the section must be avoided. Precise timing of this procedure depends on the thickness of the section and the glass as well as on the temperature of the cryostate and the hot plate. Short practice suffices to obtain flat sections with very few or no air bubbles (Fig. 3C, D). Two counters and an electronic “window” solved the remaining problems. The sections to be rejected are counted by a stepdown counter (Predetermining counter S-4330 by Coulboum
histochemical
for work with diffusible substances. During our work with a Bright (Hacker in USA) cryostat (the least expensive among those able to cut blocks of the size of the monkey brain) we encountered several problems: First, sections from large brains are notoriously difficult to mount. Considerable patience and dexterity are needed in order to avoid wrinkles or trapped air bubbles. Hand’s (I) solution was to divide large brains into blocks to be cut separately. Secondly, in order to keep constant the distance between saved sections, the technician had to count the sections to be discarded. Any distraction may result in varying distance between mounted sections. Thirdly, the block-advancing mechanism in Bright microtome must be reset after a number of sections is cut. If this count is not precise one must either reset the microtome more often than necessary or risk a time-consuming rewinding of a loose screw. Fourthly, we were wasting time while waiting for a small block to descend slowly along the whole path. Finally, we found that the antiroll plate and the knife-extension plate provided by the manufacturer were inconvenient. Several modifications of the cryostat (model 1980) and a new method of mounting the sections eliminated the problems described above. Among the cryostat modifications, the antiroll plate was enlarged to cover almost the entire space between the knifeholders. The new plate was made of perspex and had the same dimensions, except its breadth, as the original one. The angle between the antiroll plate and the knife was reduced in order to prevent bending the sections. The knife-extension was shaped as a ski-jump ramp in order to slide large sections onto the glass (Fig. 1). The shape of the ramp can be seen in Fig. 1. The dimensions ‘Requests for reprints should be addressed Denmark.
Cryostat
to Ivan Divac, Institute of Neurophysiology.
437
Panum Institute, Blegdamsvej
3C. DK-2200 Copenhagen
N.,
,OBJECT
HOLDER
I
SKI-JUMP
BLOCK
MAL
PLATFORM
1NSULAfiON
FTG. 2. The frame and block used for mounting large sections.
FIG. 1. Modifications of the aatiroll plate and the knife-extension.
Ins~m~nts, Box 2551, Lehigh Valley, PA 18001) which is set at the number of sections to be rejected. After the p~de~~~ number of strokes the serial cutting stops automatically and the section to be mounted is cut and saved. The counter is reset, and another series of sections to be rejected is cut. A cumulative counter counts all strokes and can be used to reset the block-advancing mechanism before the screw falls off. Finally, we provided an electronic “window” which could be
set according to the size and position of the block. The purpose of the window is to have the block move at the highest speed at al# times except while the tissue is being cut. In this way much time is saved, particularly when one must cut all sections at a very low and even speed, as is required by the 2-deoxyglucose method. Although more advanced cryostats incorporate some of the functions we describe above, the diagram of the electronic circuit is given (Fig. 4) for those laboratories which have more manpower than means to buy expensive equipment.
The research which led to the present& described method was supported by the Danish Medical Research Council. We thank Kirsten Lindberg for the drawings, Ulla Mogensen for cutting tbe brains and suggesting improvements, F. Riis for help with photography, Margit L@vgreenfor typing and R. Gunilla E. &erg for comments on the manuscript.
REFERENCE I.Hand, P. J. The 2-deoxyghzcose metbrxi. In: Heimer, L.; RoBa&, M. E., eds. Neuroanatomical tract-tracing methods. New York: Plenum; 198151 l-538.
CRYOSTAT
SECTIONING
OF LARGE
BRAINS
439
FIG. 3. Autoradiograms of monkey sections mounted by the presently described method. A and B illustrate consequence of late or insufficient press of the block on the section at the mc~ment of placing on the hot plate. C and D present successfull); mounted sections. The ma~nifieations of different sections are not equal.
f
+
f
t
e
+ FIG. 4. Diagram of the speed electronic circuitry subserving the window and the counters,
