Journal of Neuroscience Methods, 1 (1979) 179--183 © Elsevier/North-Holland Biomedical Press
179
P R E P A R I N G A U T O R A D I O G R A M S OF SERIAL SECTIONS F O R E L E C T R O N MICROSCOPY
THOMAS L. DAVIS, ROBERT F. SPENCER * and PETER STERLING
Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia, Pa. 19104 (U.S.A.) (Received April 23rd, 1979) (Revised version received and accepted May 21st, 1979)
A reliable procedure is described for preparing series of up to 300 consecutive thin sections for EM autoradiography.
INTRODUCTION
The autoradiographic method for tracing axonal pathways has become popular recently for several reasons. It is capable of demonstrating patterns of axonal arborizations that go unrecognized with silver methods, and it avoids labeling axons which pass through the site of isotope injection b u t do n o t originate there. The method offers advantages at the electron microscope (EM) level as well. It labels a higl~er fraction of the synaptic population than can be identified by degeneration because at any given survival time only a small proportion of the degenerating terminals remain in contact with their postsynaptic structures. We estimate, as did LeVay and Gilbert (1976), that a b o u t three times as many lateral geniculate terminals in cortical area 17 are evident by EM autoradiography as by the degeneration method. Furthermore, it is possible with autoradiography but not with the degeneration m e t h o d to see the normal morphology of the terminals. Unfortunately, the presence of a single silver grain over a synaptic terminal in an electron micrograph does not necessarily indicate the presence of radioactivity within the terminal. The uncertainty exists because the terminals are often small (0.5--1.0 pm) and thus either below or uncomfortably close to the resolution of the m e t h o d (Salpeter et al., 1969). One way to tell whether a particular terminal is labeled without resorting to statistical techniques (Blackett and Parry, 1973) is to examine the same terminal in autoradiograms from successive sections. If the terminal is labeled in most or all of these, while adjacent terminals bear grains in only
* Present address: Department of Anatomy, Medical College of Virginia, Richmond, Va.
180 one or two sections, then it may be counted as a positive (see Fig. 3). If it were possible, furthermore, to produce long series of EM autoradiograms, one could reconstruct not merely the labeled terminals themselves, but also the patterns in which they are distributed over the surfaces of whole neurons. We describe here our procedure for producing long series of EM autoradiograms. Although it involves only a few modifications of the original "substrate" technique (Kopriwa, 1973; Salpeter and Bachmann, 1964), the small improvements render the m e t h o d reliable enough for workers in our laboratory to produce series of up to 300 autoradiograms. PREPARATION OF AUTORADIOGRAMS Injections of isotope and perfusion of the animal for electron microscopy are performed according to standard methods (Cowan et al., 1972). Fixed tissue is cut on a Vibratome in alternating thick (250 t~m) and thin (30 pm) sections. The thick sections are osmicated, stained en bloc in uranyl acetate and infiltrated with Epon. Each thick section is placed with a drop of Epon on a vinyl microscope slide (Rinzl 144, Arthur H. Thomas) and a block of cured Epon (cast from a BEEM capsule) is pressed gently on top of it. After curing, the slide peels easily from the section which is now m o u n t e d on the block. The thin sections are placed on acid-cleaned slides and prepared for light microscope autoradiography. A region of optimal labeling is first identified in a light microscope autoradiogram; a mesa is then trimmed to include the corresponding region from the adjacent thick section. Ribbons of ultrathin sections (silver--gold interference colors) are cut from the mesa and picked from the trough with a copper loop. The loop is m o u n t e d on a micromanipulator and lowered until the drop of water bearing the ribbon contacts a collodion-coated slide (Fig. 1). The water is gently aspirated via a micropipette, also m o u n t e d in a micromanipulator, until the ribbon rests on the collodion film. Careful preparation of the collodion-coated slides is crucial for stripping the film subsequent to development of the autoradiogram. Slides (frosted on one end) are soaked overnight in a saturated, aqueous solution of Alconox (Sparkleen did not work). The slides are rinsed in tap water, then deionized water, dried with Kimwipes, and blown free of debris with a Freon duster. Surgical gloves are worn while handling the wet slides to prevent their contamination by oils. The slides are dipped into 1.5% collodion in amyl acetate (Ladd Research Industries, Inc.) and hung to dry. Excess collodion collects at the b o t t o m edge of the slide forming a ridge which is used later as a handle in stripping. A sample film, when stripped and cast on water, should be purple. Such a sturdy film is an advantage in subsequent handling and presents no impediment to the electron beam because it is dissolved before that stage is reached. A collodion-coated slide, now bearing a ribbon, is coated in an evaporator with a thin layer of carbon (roughly 10 rim). Excessive heat at this stage will
181
section "ribbon" ....
~ ' ~
/
micr°pipefte
water
;e
slide Fig. 1. Transfer of sections from loop to collodion-eoate~ slide.
bake the film to the slide and make subsequent stripping difficult. We avoid this by placing the slides as far from the arc as possible and by sharpening one of the carbon rods to a fine point. Emulsion (Ilford L4) is diluted (1 part emulsion to 3 parts deionized water) and maintained at 40°C in a water bath. The slides are coated one at a time with a gold--purple film of emulsion using a variable speed dipping machine. The mechanical dipper minimizes variation in emulsion thickness caused by vibration and by variations in the rate at which the slide is withdrawn from the emulsion (Kopriwa, 1967). The thickness of the emulsion can be altered simply by changing the rate of withdrawal -- the slower the speed, the thinner the film. The slides are dried for several minutes, placed in light tight boxes containing dessicant, wrapped in foil, and exposed at 4°C. The autoradiograms are developed for 2 min in D-19 at 19°C, rinsed for 15 sec in deionized water, fixed for 5 min in Kodak Rapid Fixer (no hardener) and rinsed again for at least 5 min. During these procedures the slides
Formvorcoote grdII Car0oo h__.
c,,o0 f/f/
Woter
Fig. 2. Mounting formvar-coated grid on developed autoradiogram.
182 are held f r o s t e d end u p a n d the levels o f the solutions are m a i n t a i n e d b e l o w t h e u p p e r margin o f t h e c o l l o d i o n film. T h e film is r e a d y to be s t r i p p e d w h e n b u b b l e s f o r m b e t w e e n it a n d the glass slide. T h e first step in s t r i p p i n g is to r e m o v e m o s t o f t h e w a t e r f r o m the film b y b l o w i n g gently across it w i t h a F r e o n duster. We t h e n scrape the sides and b o t t o m o f t h e slide w i t h a clean r a z o r blade and p u s h the ridge o f c o l l o d i o n f r o m the b o t t o m o f the slide t o w a r d the f r o s t e d end. T h e slide is t h e n m o u n t e d on a m i c r o m a n i p u l a t o r and l o w e r e d i n t o a t r o u g h o f d e i o n i z e d
Fig. 3. Four serial electron microscope autoradiograms of synaptic terminals in cat lateral geniculate nucleus. F, terminals, labeled by [3H]gamma-aminobutyric acid, are postsynaptic to retinal terminal (RLP). (Accelerating voltage 120 kV.) Scale = 0.5 pm.
183
water at an angle of 45 ° (Fig. 2). The film separates from the slide as it is lowered into the water. If patches of the film adhere to the slide during this procedure they are teased free b y gently tugging with a sharp probe at the thickened end of the film. Stripping is terminated when the portion of the film bearing the ribbon has floated free but the upper margin of the film is still attached to the slide. A formvar-coated slot grid held by a vacuum forceps is positioned over the ribbon with a micromanipulator, lowered to contact the ribbon, and released. The collodion film is then scored near its upper margin and floated free by lowering the slide. The film, bearing the ribbon and grid, is removed from the trough by placing a piece of lens tissue on top of it and lifting slowly. The grid is excised from the surrounding film, placed in a staining ring, and soaked for 15 min in each of three changes of amyl acetate to remove the collodion and expose the upper surface of the section. When the grid is dry, it is ready to be stained (30 min, 4% methanolic uranyl acetate; 1 min, 1% aqueous lead citrate) and viewed in the electron microscope. The final autoradiogram is multilayered (formvar, emulsion, carbon, and section) and presents a formidable obstacle to the electron beam. In our experience accelerating voltages of 100 or 120 kV do less damage to the autoradiogram and provide crisper images than do lower voltages (50--80 kV) (Fig. 3). ACKNOWLEDGEMENTS
We thank Dr. Pierluigi Gambetti for early instruction in EM autoradiographic technique and Mr. Fred Letterio and Mr. Ed Shalna for design and construction of the mechanical dipper. Supported by NEI Grants R01-00828 and EY01583. REFERENCES Blackett, N.M. and Parry, D.M. (1973) A new method for analyzing electron microscope autoradiographs using hypothetical grain distributions, J. Cell Biol., 59: 9. Cowan, W.M., Gottlieb, D.I., Hendrickson, A.E., Price, J.L. and Woolsey, T.A. (1972) The autoradiographic demonstration of axonal connections in the central nervous system, Brain Res., 37 : 21--51. Kopriwa, B.M. (1967) A semiautomatic instrument for the radioautographic coating technique, J. Histochem. Cytochem., 14: 923--928. Kopriwa, B.M. (1973) A reliable, standardized method for ultrastructural electron microscopic radioautography, J. Histochemie, 37: 1--17. LeVat, S. and Gilbert, C.D. (1976) Laminar patterns of geniculocortical projection in the cat, Brain Res., 112: 1--16. Salpeter, M.M. and Bachmann, L. (1964) Autoradiography with the electron microscope. I. A procedure for improving resolution, sensitivity and contrast, J. Cell Biol., 22: 469--477. Salpeter, M.M., Bachmann, L. and Salpeter, E.E. (1969) Resolution in electron microscope radioautography, J. Cell Biol., 41: 1--20.
179
P R E P A R I N G A U T O R A D I O G R A M S OF SERIAL SECTIONS F O R E L E C T R O N MICROSCOPY
THOMAS L. DAVIS, ROBERT F. SPENCER * and PETER STERLING
Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia, Pa. 19104 (U.S.A.) (Received April 23rd, 1979) (Revised version received and accepted May 21st, 1979)
A reliable procedure is described for preparing series of up to 300 consecutive thin sections for EM autoradiography.
INTRODUCTION
The autoradiographic method for tracing axonal pathways has become popular recently for several reasons. It is capable of demonstrating patterns of axonal arborizations that go unrecognized with silver methods, and it avoids labeling axons which pass through the site of isotope injection b u t do n o t originate there. The method offers advantages at the electron microscope (EM) level as well. It labels a higl~er fraction of the synaptic population than can be identified by degeneration because at any given survival time only a small proportion of the degenerating terminals remain in contact with their postsynaptic structures. We estimate, as did LeVay and Gilbert (1976), that a b o u t three times as many lateral geniculate terminals in cortical area 17 are evident by EM autoradiography as by the degeneration method. Furthermore, it is possible with autoradiography but not with the degeneration m e t h o d to see the normal morphology of the terminals. Unfortunately, the presence of a single silver grain over a synaptic terminal in an electron micrograph does not necessarily indicate the presence of radioactivity within the terminal. The uncertainty exists because the terminals are often small (0.5--1.0 pm) and thus either below or uncomfortably close to the resolution of the m e t h o d (Salpeter et al., 1969). One way to tell whether a particular terminal is labeled without resorting to statistical techniques (Blackett and Parry, 1973) is to examine the same terminal in autoradiograms from successive sections. If the terminal is labeled in most or all of these, while adjacent terminals bear grains in only
* Present address: Department of Anatomy, Medical College of Virginia, Richmond, Va.
180 one or two sections, then it may be counted as a positive (see Fig. 3). If it were possible, furthermore, to produce long series of EM autoradiograms, one could reconstruct not merely the labeled terminals themselves, but also the patterns in which they are distributed over the surfaces of whole neurons. We describe here our procedure for producing long series of EM autoradiograms. Although it involves only a few modifications of the original "substrate" technique (Kopriwa, 1973; Salpeter and Bachmann, 1964), the small improvements render the m e t h o d reliable enough for workers in our laboratory to produce series of up to 300 autoradiograms. PREPARATION OF AUTORADIOGRAMS Injections of isotope and perfusion of the animal for electron microscopy are performed according to standard methods (Cowan et al., 1972). Fixed tissue is cut on a Vibratome in alternating thick (250 t~m) and thin (30 pm) sections. The thick sections are osmicated, stained en bloc in uranyl acetate and infiltrated with Epon. Each thick section is placed with a drop of Epon on a vinyl microscope slide (Rinzl 144, Arthur H. Thomas) and a block of cured Epon (cast from a BEEM capsule) is pressed gently on top of it. After curing, the slide peels easily from the section which is now m o u n t e d on the block. The thin sections are placed on acid-cleaned slides and prepared for light microscope autoradiography. A region of optimal labeling is first identified in a light microscope autoradiogram; a mesa is then trimmed to include the corresponding region from the adjacent thick section. Ribbons of ultrathin sections (silver--gold interference colors) are cut from the mesa and picked from the trough with a copper loop. The loop is m o u n t e d on a micromanipulator and lowered until the drop of water bearing the ribbon contacts a collodion-coated slide (Fig. 1). The water is gently aspirated via a micropipette, also m o u n t e d in a micromanipulator, until the ribbon rests on the collodion film. Careful preparation of the collodion-coated slides is crucial for stripping the film subsequent to development of the autoradiogram. Slides (frosted on one end) are soaked overnight in a saturated, aqueous solution of Alconox (Sparkleen did not work). The slides are rinsed in tap water, then deionized water, dried with Kimwipes, and blown free of debris with a Freon duster. Surgical gloves are worn while handling the wet slides to prevent their contamination by oils. The slides are dipped into 1.5% collodion in amyl acetate (Ladd Research Industries, Inc.) and hung to dry. Excess collodion collects at the b o t t o m edge of the slide forming a ridge which is used later as a handle in stripping. A sample film, when stripped and cast on water, should be purple. Such a sturdy film is an advantage in subsequent handling and presents no impediment to the electron beam because it is dissolved before that stage is reached. A collodion-coated slide, now bearing a ribbon, is coated in an evaporator with a thin layer of carbon (roughly 10 rim). Excessive heat at this stage will
181
section "ribbon" ....
~ ' ~
/
micr°pipefte
water
;e
slide Fig. 1. Transfer of sections from loop to collodion-eoate~ slide.
bake the film to the slide and make subsequent stripping difficult. We avoid this by placing the slides as far from the arc as possible and by sharpening one of the carbon rods to a fine point. Emulsion (Ilford L4) is diluted (1 part emulsion to 3 parts deionized water) and maintained at 40°C in a water bath. The slides are coated one at a time with a gold--purple film of emulsion using a variable speed dipping machine. The mechanical dipper minimizes variation in emulsion thickness caused by vibration and by variations in the rate at which the slide is withdrawn from the emulsion (Kopriwa, 1967). The thickness of the emulsion can be altered simply by changing the rate of withdrawal -- the slower the speed, the thinner the film. The slides are dried for several minutes, placed in light tight boxes containing dessicant, wrapped in foil, and exposed at 4°C. The autoradiograms are developed for 2 min in D-19 at 19°C, rinsed for 15 sec in deionized water, fixed for 5 min in Kodak Rapid Fixer (no hardener) and rinsed again for at least 5 min. During these procedures the slides
Formvorcoote grdII Car0oo h__.
c,,o0 f/f/
Woter
Fig. 2. Mounting formvar-coated grid on developed autoradiogram.
182 are held f r o s t e d end u p a n d the levels o f the solutions are m a i n t a i n e d b e l o w t h e u p p e r margin o f t h e c o l l o d i o n film. T h e film is r e a d y to be s t r i p p e d w h e n b u b b l e s f o r m b e t w e e n it a n d the glass slide. T h e first step in s t r i p p i n g is to r e m o v e m o s t o f t h e w a t e r f r o m the film b y b l o w i n g gently across it w i t h a F r e o n duster. We t h e n scrape the sides and b o t t o m o f t h e slide w i t h a clean r a z o r blade and p u s h the ridge o f c o l l o d i o n f r o m the b o t t o m o f the slide t o w a r d the f r o s t e d end. T h e slide is t h e n m o u n t e d on a m i c r o m a n i p u l a t o r and l o w e r e d i n t o a t r o u g h o f d e i o n i z e d
Fig. 3. Four serial electron microscope autoradiograms of synaptic terminals in cat lateral geniculate nucleus. F, terminals, labeled by [3H]gamma-aminobutyric acid, are postsynaptic to retinal terminal (RLP). (Accelerating voltage 120 kV.) Scale = 0.5 pm.
183
water at an angle of 45 ° (Fig. 2). The film separates from the slide as it is lowered into the water. If patches of the film adhere to the slide during this procedure they are teased free b y gently tugging with a sharp probe at the thickened end of the film. Stripping is terminated when the portion of the film bearing the ribbon has floated free but the upper margin of the film is still attached to the slide. A formvar-coated slot grid held by a vacuum forceps is positioned over the ribbon with a micromanipulator, lowered to contact the ribbon, and released. The collodion film is then scored near its upper margin and floated free by lowering the slide. The film, bearing the ribbon and grid, is removed from the trough by placing a piece of lens tissue on top of it and lifting slowly. The grid is excised from the surrounding film, placed in a staining ring, and soaked for 15 min in each of three changes of amyl acetate to remove the collodion and expose the upper surface of the section. When the grid is dry, it is ready to be stained (30 min, 4% methanolic uranyl acetate; 1 min, 1% aqueous lead citrate) and viewed in the electron microscope. The final autoradiogram is multilayered (formvar, emulsion, carbon, and section) and presents a formidable obstacle to the electron beam. In our experience accelerating voltages of 100 or 120 kV do less damage to the autoradiogram and provide crisper images than do lower voltages (50--80 kV) (Fig. 3). ACKNOWLEDGEMENTS
We thank Dr. Pierluigi Gambetti for early instruction in EM autoradiographic technique and Mr. Fred Letterio and Mr. Ed Shalna for design and construction of the mechanical dipper. Supported by NEI Grants R01-00828 and EY01583. REFERENCES Blackett, N.M. and Parry, D.M. (1973) A new method for analyzing electron microscope autoradiographs using hypothetical grain distributions, J. Cell Biol., 59: 9. Cowan, W.M., Gottlieb, D.I., Hendrickson, A.E., Price, J.L. and Woolsey, T.A. (1972) The autoradiographic demonstration of axonal connections in the central nervous system, Brain Res., 37 : 21--51. Kopriwa, B.M. (1967) A semiautomatic instrument for the radioautographic coating technique, J. Histochem. Cytochem., 14: 923--928. Kopriwa, B.M. (1973) A reliable, standardized method for ultrastructural electron microscopic radioautography, J. Histochemie, 37: 1--17. LeVat, S. and Gilbert, C.D. (1976) Laminar patterns of geniculocortical projection in the cat, Brain Res., 112: 1--16. Salpeter, M.M. and Bachmann, L. (1964) Autoradiography with the electron microscope. I. A procedure for improving resolution, sensitivity and contrast, J. Cell Biol., 22: 469--477. Salpeter, M.M., Bachmann, L. and Salpeter, E.E. (1969) Resolution in electron microscope radioautography, J. Cell Biol., 41: 1--20.
