American Journal of Hematology 39:223-225 (1992)
Application of Immunocryoultramicrotomy to Free Cells: Ultrastructural Localization of Myeloperoxidase in Neutrophils of Human Peripheral Blood Nagahito Saito, Nobuo Takemori, Katsuyuki Hirai, Noriko Kubota, Ryuichi Onodera, and Masayoshi Namiki Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Hokkaido, Japan
lmmunocryoultramicrotomy is generally considered the most suitable method for observing the immunostainings by monoclonal antibodies to intracellular antigens by electron microscopy. However, this procedure with the free cells such as peripheral blood cells or cultured cells is not widely used because of the difficulty in preparing ultrathin frozen sections. We report here a new technique for making ultrathin frozen sections of peripheral blood cells by positioning the cell in a semigelatinized medium for ultrasectioning. Monoclonal antibody against rnyeloperoxidase(MPO) was reacted with the ultrathin sections made with this new technique to observe the ultrastructural localization of intracellular MPO. MPO was observed in the primary granules of the neutrophils. Key words: cryoultramicrotomy, ultrathin frozen section, immunogold staining, MPO
INTRODUCTION
Myeloperoxidase (MPO) is known to be contained in various blood cells such as neutrophils, monocytes, and eosinophils. In neutrophils, MPO is detected in the primary granules by ultracytochemical techniques [ 1,2]. Immunogold staining is a very effective method for observing ultrastructural localization of antigens [3]. However, it is very difficult to detect intracellular antigens with monoclonal antibodies using postembedding methods [4]. Recently, a new method [5,6] in immunoelectron microscopy known as immunocryoultramicrotomy, which has the advantages of antigenicity and morphological preservation, has been reported. However, there are few reports for free cells due to the technical difficulty in making ultrathin frozen sections. In this paper, we describe an improved method of immunocryoultramicrotomy for free cells and demonstrate the localization of MPO in human neutrophils using monoclonal antibody against human MPO. MATERIALS AND METHODS
Buffy coats of peripheral blood cells from six normal volunteers were fixed in 0.5% glutaraldehyde 2% paraformaldehyde 0.32%picric acid 1.5% sucrose 0.05 M phosphate buffer (pH 7.4) for 30 min at 4°C. After 0 1992 Wiley-Liss, Inc.
washing with 0.1 M phosphate buffer (pH 7.4), the fixed specimens were suspended in a mixture of human plasma and 0.02 M CaC1, solution (v/v; 2:l). Then, thrombin (100 U/ml), in a volume equal to that of CaCI,, was added into the cell suspension to semigelatinize the plasma containing fixed free cells. After centrifugation, the cell pellet was fixed in 0.5% paraformaldehyde 8% sucrose 0.05 M phosphate buffer (pH 7.4) for 24 hours at 4°C and stored in 2.3 M sucrose 0.1 M phosphate buffer (pH 7.4) for a few days before cryosectioning. The specimen in a small block was mounted on a specimen carrier and was rapidly frozen in liquid nitrogen. Then, ultrathin frozen sections were made using a cryosectioning system (Ultracut FC4E, Reichert-Jung) at -95°C. The ultrathin frozen sections were mounted on the grids and immunostained as previously reported [6]. Briefly, the ultrathin frozen sections were placed onto a Formvar-carboncoated grid using a droplet of the solution containing sucrose, gelatin, and glycerol. The grid was floated on 0.1 % glutaraldehyde 0.05 M phosphate buffer (pH 7.4) Received for publication August 7, 1991; accepted September 26, 1991. Address reprint requests to Nagahito Saito, MD, Third Department of Internal Medicine, Asahikawa Medical College, Nishikagura-4-5-31 1 , Asahikawa, Hokkaido, Japan.
224
Brief Report: Saito et al.
Fig. 1. a: Peripheral blood ells from a normal volunteer. lmmunostaining by monoclonal antibody against human MPO. Some neutrophils are observed in the view field. Bar = 5 Wm. b: Higher magnification of area at arrow in a. Gold particles are observed in the granules (arrowheads). Bar = 0.5 Fm.
to glue the sections onto the grid. After washing with 0.1 M phosphate-buffered saline (PBS; pH 7.4), the grid was floated on 1% normal goat serum and then reacted with appropriately diluted monoclonal antibody against human MPO (Dakopatts) for 10-12 hr at 4°C. Goat antiserum specific for mouse IgG conjugated with 10 nm gold particles (Gam GIO; Janssen) was used as the secondary antibody. The sections were fixed again with 2% glutaraldehyde 0.05 M phosphate buffer (pH 7.4) and then with 2% O,O, 0.1 M PBS (pH 7.4) before embedding in Spurr epoxy resin (TAAB). After polymerization, the sections were stained with both uranyl acetate and lead citrate and observed via electron microscopy. As a control, primary antibody was omitted from the procedures described above. RESULTS
Using our method, ultrathin frozen sections could easily be made by cryosectioning (Fig. la). Ultrastructural localization of MPO was demonstrated by the observa-
tion of the 10 nm gold particles. The gold particles were observed in the larger granules, which were considered to be the primary granules. No gold particles were observed in other cellular organelles except for the granules (Fig. Ib). In control sections, no gold particles were observed. DISCUSSION
Recent advances in cryosectioning have made it possible to make ultrathin frozen sections from parenchymatous tissues without much difficulty. Nevertheless, it is very difficult to make ultrathin frozen sections from free cells such as peripheral blood cells or cultured cells with conventional cryoultramicrotomy. We thus developed a modified method for the cryosectioning of the free cell specimens; the fixed peripheral blood cells were semigelatinized in plasma, calcium solution, and thrombin at a ratio of 2: I :1 . This process provided the cell specimens with suitable viscosity for cryosectioning and made it possible to recover the ultrathin frozen sections. This
Brief Report: MPO in Neutrophils
modified method is expected to be a powerful technique for cryosectioning of free cells. It is generally considered that MPO is synthesized in the primary granules in neutrophils [7]. This was confirmed by our modified method of immunocryoultramicrotomy, in which MPO was detected in the primary granules in neutrophils using monoclonal antibody. In immature neutrophils, MPO is also reportedly detected in the perinuclear space, rough endoplasmic reticulum, and Golgi apparatus [ 11. Whether MPO is present in those organelles of immature neutrophils warrants future investigation via immunocryoultramicrotomy . Monoclonal antibody against MPO is effective in diagnosing myeloid leukemias [81. Ultrastructural examinations of monoclonal antibody against MPO by immunocryoultramicrotomy could shed light on the nature of the leukemia cells in myeloid linage. ACKNOWLEDGMENTS We thank Mr. Miyagawa and Miss Yamamoto (Asahikawa Medical College) for technical support and Dr. Ooi (Faculty of Veterinary Medicine, Hokkaido University) for helpful discussions.
225
REFERENCES 1. Bainton DF, Ullyot JL, Farquhar MG: The development of neutrophilic polymorphonuclear leukocytes in human bone marrow. Origin and content of azurophil and specific granules. J Exp Med 134:903, 1971. 2. Graham RC, Kamovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14:291, 1966. 3. Saito N: Acute myelomonocytic leukemia: An immunoelectron rnicroscopic study. Am J Hematol 35:238, 1990. 4. Saito N, Pulford KAF, Breton-Gorius J, Mason DY, Cramer EM: Ultrastructural localization of the CD68 macrophage-associated antigens in human blood neutrophils and monocytes. Am J Pathol 139:1053, 1991. 5. Tokuyasu KT: Present state of immunocryoultramicrotomy . J Histochem Cytochem 31:164, 1983. 6. Takemori N, Hirai K, Ishikawa Y, Yamada H, Onodera R, Kumei Y, Namiki M: Immunocryoultramicrotomy: Its drawbacks and improvement. J Clin Electron Microsc 23:5, 1990. 7. Fouret P, du Bois RM, Bernaudin JF, Takahashi H, Ferrans VJ, Crystal RG: Expression of the neutrophil elastase gene during human bone marrow cell differentiation. J Exp Med 169833, 1989. 8. Dijkstra K, Kluin-Nelemans HC: No difference between cytochemical and immunological detection of myeloperoxidase in AML. Br J Haemato1 75:630, 1990.
Application of Immunocryoultramicrotomy to Free Cells: Ultrastructural Localization of Myeloperoxidase in Neutrophils of Human Peripheral Blood Nagahito Saito, Nobuo Takemori, Katsuyuki Hirai, Noriko Kubota, Ryuichi Onodera, and Masayoshi Namiki Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Hokkaido, Japan
lmmunocryoultramicrotomy is generally considered the most suitable method for observing the immunostainings by monoclonal antibodies to intracellular antigens by electron microscopy. However, this procedure with the free cells such as peripheral blood cells or cultured cells is not widely used because of the difficulty in preparing ultrathin frozen sections. We report here a new technique for making ultrathin frozen sections of peripheral blood cells by positioning the cell in a semigelatinized medium for ultrasectioning. Monoclonal antibody against rnyeloperoxidase(MPO) was reacted with the ultrathin sections made with this new technique to observe the ultrastructural localization of intracellular MPO. MPO was observed in the primary granules of the neutrophils. Key words: cryoultramicrotomy, ultrathin frozen section, immunogold staining, MPO
INTRODUCTION
Myeloperoxidase (MPO) is known to be contained in various blood cells such as neutrophils, monocytes, and eosinophils. In neutrophils, MPO is detected in the primary granules by ultracytochemical techniques [ 1,2]. Immunogold staining is a very effective method for observing ultrastructural localization of antigens [3]. However, it is very difficult to detect intracellular antigens with monoclonal antibodies using postembedding methods [4]. Recently, a new method [5,6] in immunoelectron microscopy known as immunocryoultramicrotomy, which has the advantages of antigenicity and morphological preservation, has been reported. However, there are few reports for free cells due to the technical difficulty in making ultrathin frozen sections. In this paper, we describe an improved method of immunocryoultramicrotomy for free cells and demonstrate the localization of MPO in human neutrophils using monoclonal antibody against human MPO. MATERIALS AND METHODS
Buffy coats of peripheral blood cells from six normal volunteers were fixed in 0.5% glutaraldehyde 2% paraformaldehyde 0.32%picric acid 1.5% sucrose 0.05 M phosphate buffer (pH 7.4) for 30 min at 4°C. After 0 1992 Wiley-Liss, Inc.
washing with 0.1 M phosphate buffer (pH 7.4), the fixed specimens were suspended in a mixture of human plasma and 0.02 M CaC1, solution (v/v; 2:l). Then, thrombin (100 U/ml), in a volume equal to that of CaCI,, was added into the cell suspension to semigelatinize the plasma containing fixed free cells. After centrifugation, the cell pellet was fixed in 0.5% paraformaldehyde 8% sucrose 0.05 M phosphate buffer (pH 7.4) for 24 hours at 4°C and stored in 2.3 M sucrose 0.1 M phosphate buffer (pH 7.4) for a few days before cryosectioning. The specimen in a small block was mounted on a specimen carrier and was rapidly frozen in liquid nitrogen. Then, ultrathin frozen sections were made using a cryosectioning system (Ultracut FC4E, Reichert-Jung) at -95°C. The ultrathin frozen sections were mounted on the grids and immunostained as previously reported [6]. Briefly, the ultrathin frozen sections were placed onto a Formvar-carboncoated grid using a droplet of the solution containing sucrose, gelatin, and glycerol. The grid was floated on 0.1 % glutaraldehyde 0.05 M phosphate buffer (pH 7.4) Received for publication August 7, 1991; accepted September 26, 1991. Address reprint requests to Nagahito Saito, MD, Third Department of Internal Medicine, Asahikawa Medical College, Nishikagura-4-5-31 1 , Asahikawa, Hokkaido, Japan.
224
Brief Report: Saito et al.
Fig. 1. a: Peripheral blood ells from a normal volunteer. lmmunostaining by monoclonal antibody against human MPO. Some neutrophils are observed in the view field. Bar = 5 Wm. b: Higher magnification of area at arrow in a. Gold particles are observed in the granules (arrowheads). Bar = 0.5 Fm.
to glue the sections onto the grid. After washing with 0.1 M phosphate-buffered saline (PBS; pH 7.4), the grid was floated on 1% normal goat serum and then reacted with appropriately diluted monoclonal antibody against human MPO (Dakopatts) for 10-12 hr at 4°C. Goat antiserum specific for mouse IgG conjugated with 10 nm gold particles (Gam GIO; Janssen) was used as the secondary antibody. The sections were fixed again with 2% glutaraldehyde 0.05 M phosphate buffer (pH 7.4) and then with 2% O,O, 0.1 M PBS (pH 7.4) before embedding in Spurr epoxy resin (TAAB). After polymerization, the sections were stained with both uranyl acetate and lead citrate and observed via electron microscopy. As a control, primary antibody was omitted from the procedures described above. RESULTS
Using our method, ultrathin frozen sections could easily be made by cryosectioning (Fig. la). Ultrastructural localization of MPO was demonstrated by the observa-
tion of the 10 nm gold particles. The gold particles were observed in the larger granules, which were considered to be the primary granules. No gold particles were observed in other cellular organelles except for the granules (Fig. Ib). In control sections, no gold particles were observed. DISCUSSION
Recent advances in cryosectioning have made it possible to make ultrathin frozen sections from parenchymatous tissues without much difficulty. Nevertheless, it is very difficult to make ultrathin frozen sections from free cells such as peripheral blood cells or cultured cells with conventional cryoultramicrotomy. We thus developed a modified method for the cryosectioning of the free cell specimens; the fixed peripheral blood cells were semigelatinized in plasma, calcium solution, and thrombin at a ratio of 2: I :1 . This process provided the cell specimens with suitable viscosity for cryosectioning and made it possible to recover the ultrathin frozen sections. This
Brief Report: MPO in Neutrophils
modified method is expected to be a powerful technique for cryosectioning of free cells. It is generally considered that MPO is synthesized in the primary granules in neutrophils [7]. This was confirmed by our modified method of immunocryoultramicrotomy, in which MPO was detected in the primary granules in neutrophils using monoclonal antibody. In immature neutrophils, MPO is also reportedly detected in the perinuclear space, rough endoplasmic reticulum, and Golgi apparatus [ 11. Whether MPO is present in those organelles of immature neutrophils warrants future investigation via immunocryoultramicrotomy . Monoclonal antibody against MPO is effective in diagnosing myeloid leukemias [81. Ultrastructural examinations of monoclonal antibody against MPO by immunocryoultramicrotomy could shed light on the nature of the leukemia cells in myeloid linage. ACKNOWLEDGMENTS We thank Mr. Miyagawa and Miss Yamamoto (Asahikawa Medical College) for technical support and Dr. Ooi (Faculty of Veterinary Medicine, Hokkaido University) for helpful discussions.
225
REFERENCES 1. Bainton DF, Ullyot JL, Farquhar MG: The development of neutrophilic polymorphonuclear leukocytes in human bone marrow. Origin and content of azurophil and specific granules. J Exp Med 134:903, 1971. 2. Graham RC, Kamovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14:291, 1966. 3. Saito N: Acute myelomonocytic leukemia: An immunoelectron rnicroscopic study. Am J Hematol 35:238, 1990. 4. Saito N, Pulford KAF, Breton-Gorius J, Mason DY, Cramer EM: Ultrastructural localization of the CD68 macrophage-associated antigens in human blood neutrophils and monocytes. Am J Pathol 139:1053, 1991. 5. Tokuyasu KT: Present state of immunocryoultramicrotomy . J Histochem Cytochem 31:164, 1983. 6. Takemori N, Hirai K, Ishikawa Y, Yamada H, Onodera R, Kumei Y, Namiki M: Immunocryoultramicrotomy: Its drawbacks and improvement. J Clin Electron Microsc 23:5, 1990. 7. Fouret P, du Bois RM, Bernaudin JF, Takahashi H, Ferrans VJ, Crystal RG: Expression of the neutrophil elastase gene during human bone marrow cell differentiation. J Exp Med 169833, 1989. 8. Dijkstra K, Kluin-Nelemans HC: No difference between cytochemical and immunological detection of myeloperoxidase in AML. Br J Haemato1 75:630, 1990.
