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En face stripping of vascular endothelium
MICROVASCULAR
RESEARCH,
4,77-80 (1972)
En Face Stripping ROBERT
of Vascular
Endothelium
M. SADE AND JUDAH FOLKMAN
The Department of Surgery, Harvard Medical School, and The Children’s Hospital Medical Center, Boston, Massachusetts 021IS Received August 5, 1971 A technique for removing the endothelium from the intimal surface of a major vessel as a flat sheet is presented. The method is particularly suitable for autoradiography of endothelium.
The study of endothelium as a flat sheetrather than in fixed tissue sections has found application in such investigations of endothelium as its microscopic anatomy (l), its responsetoinjury(5),itsregenerationasmeasuredbyautoradiography(4),anditsenzyme activity determined histochemically(8). The great advantage of en facepreparations over standard histologic sections is the very large number of cells that can be studied in one specimen, on the order of tens of thousands. Our interest in techniques of producing en face preparations of major vesselendothelium stemsfrom our search for an in vitro assayfor tumor angiogenesisfactor (TAF). TAF, a factor that is mitogenic to endothelium, was recently isolated from tumor cells in this laboratory (2). The methods used to obtain endothelial sheetshave included surgical dissection (3) freezing the vessel to a glass slide (8) or to a glass slide coated with glycerine albumin fixative (4), and sealing thevesselto nitrocellulose (5), to cellulose acetatepaper (6), or to gelatin (7). Problems such as losing cells from the surface of the slide and wrinkling of nitrocellulose render most of these methods poorly suited to autoradiography. Though Obaze’s technique (4) was specifically adapted for autoradiography, we have had no experience with it. The following method for obtaining single cell thickness sheets of endothelium is rapid, simple, and has worked very well in producing autoradiographs by our rapid scintillation method (9). METHOD 1. The following preliminary steps should be carried out: a. 0.2 ml of 5 ‘A Bacto-Gelatin (Difco) is spread over a 2 cm square area on a glass slide that has been previously cleaned with absolute ethanol. The slide is dried in an oven at 80°C for at least 12 hr. b. The slide should be stored in the oven until about 15 min prior to use when it is brought to 4°C. c. Two Coplin jars, one with 10% formalin, the other with tap water, should also be kept at 4°C. 2. The vesselto be studied (we have used rat aorta) is gently irrigated with 0.15 normal saline or tissue culture medium (Medium 199). 0 1972 by Academic
Press,
Inc.
77
78
SADE AND FOLKMAN
3. It is then incised in its long axis with fine sharp dissecting scissorsand opened flat, like a postage stamp, with the adventitial side resting on a paper towel. 4. After one min of drying, the vesselon the towel is placed in 10% formalin and fixed in a flat plane. Fixation is continued for at least 2 hr, preferably overnight. 5. Following formalin fixation, the vesselis soaked in tap water for at least 30 min. 6. It is stained with Weigert’s iron-hematoxylin for 3 min, then rinsed in water. 7. The specimen is placed endothelium-up on a paper towel and very gently blotted with tissue paper so that no grossly visible water remains on the surface, then allowed to dry in room air for exactly 2 min. 8. A gelatin slide is removed from the refrigerator, the vesselimmediately placed on the slide so that the endothelium is in intimate contact with the gelatin, the vesselgently but firmly compressed on the gelatin with a firm rod (a bent paper clip serves this purpose well), and the slide with adherent vessel placed immediately into the Coplin jar with cold formalin. If ambient humidity is very high, fogging of the slide may result when it is removed from the refrigerator; if this occurs, it may be necessaryto carry out step 8 in a cold room (4°C). 9. After 30 min in cold formalin, the slide is rinsed in a second Coplin jar with cold water. 10. The slide is immersed in 95% ethanol in a petri dish at room temperature for exactly 5 min. 11. With the slide still under ethanol, a corner of the vesselis grasped with fine forceps and is gently peeled from the gelatin, leaving the endothelium as a single layer on the gelatin. Small strips of smooth muscle may also remain on the gelatin. These can be removed either with fine forceps or by gently wiping with a small cotton pledget. It is helpful to carry out this step under a dissecting microscope (10-40x). RESULTS AND DISCUSSION During the past year, over 600 strippings have beendone using this method. We have been able fairly consistently to recover X0-95 % of the endothelium present in the vessel (Fig. l), although one out of every four or five specimensmust be discarded because the endothelium has remained almost entirely with the vessel after stripping. Silver staining carried out by Poole’s method (5) has demonstrated that the cells seenon the slide have the tesselatedappearanceof endothelium. The method has proved eminently satisfactory for autoradiography. By a technique described elsewhere(9), the aorta of a rat was exposed in vitro to 3H-thymidine for 6 hr and the vessselstripped by the above technique. Rapid autoradiography was carried out by coating the gelatin slide with photographic emulsion, placing it in scintillation counting fluid for 20 hr, and developing it (9). The resulting autoradiograph is shown in Fig. 2. Although we have not used it for this purpose, this stripping technique might also be useful for obtaining living cells, by skipping from step 3 to step 7 and at step 9, instead of placing the slide in formalin, placing it in tissue culture fluid. The gelatin
EN FACE STRIPPING
79
FIG. 1. This is a sheet of endothelium from a rat’s aorta. The nuclei were stained with hematoxylin; no cytoplasmic stain was used. Note the small strip of smooth muscle in the lower left corner. This is as complete a stripping as the technique can produce (37.5~).
FIG. 2. This is a higher power view of a stripping similar to Fig. 1. The vessel has been removed from a rat, incubated with 3H-thymidine for 6 h, then stripped and autoradiographed (187.5x).
80
SADE AND FOLKMAN
then dissolves, and the endothelial cells may be isolated by the method described by Tsutsumi and Gore (7). REFERENCES 1. FL~REY, H. W., POOLE, J. C. F., AND MEEK, G. A. (1959). Endotheliai
Cells and “Cement”
Lines.
J. Pathol. Bacterial. 77, 625-636. 2. FOLKMAN, J., MERLER, E., ABERNATHY, C., AND WILLIAMS, G. (1971). Isolation of a Tumor Factor Responsible for Angiogenesis. J. Exp. Med. 133,275-288. 3. LAUTSCH, E. V., MCMILLAN, G. C., AND DUFF, G. 0. (1953). Techniques for the Study of the Normal and Atherosclerotic Arterial Intima from its Endotheliai Surface. Lab. Znuest.2,297-407. 4. OBAZE, E. R., AND PAYLING WRIGHT, H. (1968). A Modified Technique for Producing “en face” (Hlutchen) Preparation of Endotheiium for Radioautography. J. Atheroscler. Res. 8, 861-863. 5. POOLE, J. C. F., SANDERS, A. G., AND FLOREY, H. S. (1958). Regeneration of Aortic Endothelium. J. Pathol. Bacterial. 75, 133-143. 6. PUGATSCH, E. M. J., AND SAUNDERS, A. M. (1968). A New Technique for Making Htiutchen PreRes. 8,735-738. parations of Unfixed Aortic Endothelium. J. Atheroscl. 7. TSU~SUMI, H., ANLI GORE, I. (1969). Isolation of Living Endothelial Cells by Gelatin-film Stripping of Vascular Walls. Stain Technol. 44, 139-142. 8, WARREN, B. A. (1965). A Method for the Production of “enface” Preparations One Cell in Thickness.
J. Roy. Miscros. Sot. 84,407-413. 9. WEINGARD, D., FOLKMAN, J., AND SADE, R. M. (1971). Rapid Autoradiography Fluid. Unpublished.
Using Scintillation
RESEARCH,
4,77-80 (1972)
En Face Stripping ROBERT
of Vascular
Endothelium
M. SADE AND JUDAH FOLKMAN
The Department of Surgery, Harvard Medical School, and The Children’s Hospital Medical Center, Boston, Massachusetts 021IS Received August 5, 1971 A technique for removing the endothelium from the intimal surface of a major vessel as a flat sheet is presented. The method is particularly suitable for autoradiography of endothelium.
The study of endothelium as a flat sheetrather than in fixed tissue sections has found application in such investigations of endothelium as its microscopic anatomy (l), its responsetoinjury(5),itsregenerationasmeasuredbyautoradiography(4),anditsenzyme activity determined histochemically(8). The great advantage of en facepreparations over standard histologic sections is the very large number of cells that can be studied in one specimen, on the order of tens of thousands. Our interest in techniques of producing en face preparations of major vesselendothelium stemsfrom our search for an in vitro assayfor tumor angiogenesisfactor (TAF). TAF, a factor that is mitogenic to endothelium, was recently isolated from tumor cells in this laboratory (2). The methods used to obtain endothelial sheetshave included surgical dissection (3) freezing the vessel to a glass slide (8) or to a glass slide coated with glycerine albumin fixative (4), and sealing thevesselto nitrocellulose (5), to cellulose acetatepaper (6), or to gelatin (7). Problems such as losing cells from the surface of the slide and wrinkling of nitrocellulose render most of these methods poorly suited to autoradiography. Though Obaze’s technique (4) was specifically adapted for autoradiography, we have had no experience with it. The following method for obtaining single cell thickness sheets of endothelium is rapid, simple, and has worked very well in producing autoradiographs by our rapid scintillation method (9). METHOD 1. The following preliminary steps should be carried out: a. 0.2 ml of 5 ‘A Bacto-Gelatin (Difco) is spread over a 2 cm square area on a glass slide that has been previously cleaned with absolute ethanol. The slide is dried in an oven at 80°C for at least 12 hr. b. The slide should be stored in the oven until about 15 min prior to use when it is brought to 4°C. c. Two Coplin jars, one with 10% formalin, the other with tap water, should also be kept at 4°C. 2. The vesselto be studied (we have used rat aorta) is gently irrigated with 0.15 normal saline or tissue culture medium (Medium 199). 0 1972 by Academic
Press,
Inc.
77
78
SADE AND FOLKMAN
3. It is then incised in its long axis with fine sharp dissecting scissorsand opened flat, like a postage stamp, with the adventitial side resting on a paper towel. 4. After one min of drying, the vesselon the towel is placed in 10% formalin and fixed in a flat plane. Fixation is continued for at least 2 hr, preferably overnight. 5. Following formalin fixation, the vesselis soaked in tap water for at least 30 min. 6. It is stained with Weigert’s iron-hematoxylin for 3 min, then rinsed in water. 7. The specimen is placed endothelium-up on a paper towel and very gently blotted with tissue paper so that no grossly visible water remains on the surface, then allowed to dry in room air for exactly 2 min. 8. A gelatin slide is removed from the refrigerator, the vesselimmediately placed on the slide so that the endothelium is in intimate contact with the gelatin, the vesselgently but firmly compressed on the gelatin with a firm rod (a bent paper clip serves this purpose well), and the slide with adherent vessel placed immediately into the Coplin jar with cold formalin. If ambient humidity is very high, fogging of the slide may result when it is removed from the refrigerator; if this occurs, it may be necessaryto carry out step 8 in a cold room (4°C). 9. After 30 min in cold formalin, the slide is rinsed in a second Coplin jar with cold water. 10. The slide is immersed in 95% ethanol in a petri dish at room temperature for exactly 5 min. 11. With the slide still under ethanol, a corner of the vesselis grasped with fine forceps and is gently peeled from the gelatin, leaving the endothelium as a single layer on the gelatin. Small strips of smooth muscle may also remain on the gelatin. These can be removed either with fine forceps or by gently wiping with a small cotton pledget. It is helpful to carry out this step under a dissecting microscope (10-40x). RESULTS AND DISCUSSION During the past year, over 600 strippings have beendone using this method. We have been able fairly consistently to recover X0-95 % of the endothelium present in the vessel (Fig. l), although one out of every four or five specimensmust be discarded because the endothelium has remained almost entirely with the vessel after stripping. Silver staining carried out by Poole’s method (5) has demonstrated that the cells seenon the slide have the tesselatedappearanceof endothelium. The method has proved eminently satisfactory for autoradiography. By a technique described elsewhere(9), the aorta of a rat was exposed in vitro to 3H-thymidine for 6 hr and the vessselstripped by the above technique. Rapid autoradiography was carried out by coating the gelatin slide with photographic emulsion, placing it in scintillation counting fluid for 20 hr, and developing it (9). The resulting autoradiograph is shown in Fig. 2. Although we have not used it for this purpose, this stripping technique might also be useful for obtaining living cells, by skipping from step 3 to step 7 and at step 9, instead of placing the slide in formalin, placing it in tissue culture fluid. The gelatin
EN FACE STRIPPING
79
FIG. 1. This is a sheet of endothelium from a rat’s aorta. The nuclei were stained with hematoxylin; no cytoplasmic stain was used. Note the small strip of smooth muscle in the lower left corner. This is as complete a stripping as the technique can produce (37.5~).
FIG. 2. This is a higher power view of a stripping similar to Fig. 1. The vessel has been removed from a rat, incubated with 3H-thymidine for 6 h, then stripped and autoradiographed (187.5x).
80
SADE AND FOLKMAN
then dissolves, and the endothelial cells may be isolated by the method described by Tsutsumi and Gore (7). REFERENCES 1. FL~REY, H. W., POOLE, J. C. F., AND MEEK, G. A. (1959). Endotheliai
Cells and “Cement”
Lines.
J. Pathol. Bacterial. 77, 625-636. 2. FOLKMAN, J., MERLER, E., ABERNATHY, C., AND WILLIAMS, G. (1971). Isolation of a Tumor Factor Responsible for Angiogenesis. J. Exp. Med. 133,275-288. 3. LAUTSCH, E. V., MCMILLAN, G. C., AND DUFF, G. 0. (1953). Techniques for the Study of the Normal and Atherosclerotic Arterial Intima from its Endotheliai Surface. Lab. Znuest.2,297-407. 4. OBAZE, E. R., AND PAYLING WRIGHT, H. (1968). A Modified Technique for Producing “en face” (Hlutchen) Preparation of Endotheiium for Radioautography. J. Atheroscler. Res. 8, 861-863. 5. POOLE, J. C. F., SANDERS, A. G., AND FLOREY, H. S. (1958). Regeneration of Aortic Endothelium. J. Pathol. Bacterial. 75, 133-143. 6. PUGATSCH, E. M. J., AND SAUNDERS, A. M. (1968). A New Technique for Making Htiutchen PreRes. 8,735-738. parations of Unfixed Aortic Endothelium. J. Atheroscl. 7. TSU~SUMI, H., ANLI GORE, I. (1969). Isolation of Living Endothelial Cells by Gelatin-film Stripping of Vascular Walls. Stain Technol. 44, 139-142. 8, WARREN, B. A. (1965). A Method for the Production of “enface” Preparations One Cell in Thickness.
J. Roy. Miscros. Sot. 84,407-413. 9. WEINGARD, D., FOLKMAN, J., AND SADE, R. M. (1971). Rapid Autoradiography Fluid. Unpublished.
Using Scintillation