TECHNICAL NOTE |
Correspondence to: Gilbert-André Keller, Pharmacological Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. E-mail: gakeller@gene.com
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have adapted existing microwave irradiation (MWI) protocols and applied them to the processing and immunoelectron microscopy of both plastic-embedded and frozen sections. Rat livers were fixed by rapid MW irradiation in a mild fixation solution. Fixed liver tissue was either cryosectioned or dehydrated and embedded in Spurr's, Unicryl, or LR White resin. Frozen sections and sections of acrylic-embedded tissue were immunolabeled in the MW oven with an anti-catalase antibody, followed by gold labeling. Controls were processed conventionally at room temperature (RT). The use of MWI greatly shortened the fixation, processing, and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling. The higher immunogold labeling intensity was achieved after a 15-min incubation of primary antibody and gold markers under discontinued MWI at 37C. Quantification of the immunolabeling for catalase indicated a density increase of up to fourfold in the sections immunolabeled in the MW oven over that of samples immunolabeled at RT. These studies define the general conditions of fixation and immunolabeling for both acrylic resin-embedded material and frozen sections.
(J Histochem Cytochem 48:11531159, 2000)
Key Words: microwave irradiation, immunolabeling, plastic and frozen sections
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
IMMUNOELECTRON MICROSCOPY has been widely used to determine the intracellular distribution of proteins to gain information on their function. The procedure often requires a large number of samples and substantial processing time to determine unambiguously the localization of a given antigen under different experimental conditions. Postembedding methods applying microwave irradiation (MWI) to the fixation and immunolabeling of thin plastic-embedded sections have already been developed (
In our experience, frozen sections are optimal substrates to carry out immunolabeling experiments. The absence of plastic embedding medium enhances the permeation of the antibody reagents into the section, leading to a higher immunolabeling density than that achieved on plastic-embedded sections. However, immunocryoelectron microscopy is time-consuming, which is a major disadvantage because the different steps from fixation of the tissues to immunolabeling of the frozen sections are usually carried out without interruption. The technique is even more time-consuming when, after immunolabeling, the frozen sections are treated with osmium tetroxide, dehydrated, and embedded in plastic according to the ultrathin plastic embedding technique (
For the studies described here, we chose peroxisomal catalase as a model for immunolabeling because the antigen is moderately abundant and withstands fixation. Furthermore, the enzyme is mostly distributed in one subcellular compartment, and the morphological features of peroxisomes are well characterized (reviewed by
Although much has already been written about the parameters necessary for application of MW techniques to the processing and immunolabeling of samples, we recommend the articles by
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The experiments reported here were carried out in a programmable model 3450 microwave oven that allows greater temperature and energy control (Ted Pella; Redding, CA). Maintaining the temperature between 25C and 42C gives the best results without damaging the samples (
MWI Fixation of Liver Samples for both Plastic Embedding and Cryoultramicrotomy
All housing and husbandry methods were in accordance with the 1996 Guide for the Care and Use of Laboratory Animals. Livers from male rats were cut into 1-mm3 blocks that were rapidly transferred to Eppendorff tubes containing 3% formaldehyde and 0.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. The samples were placed in crushed ice to lower the temperature to 8C, removed from ice, and transferred to the MW oven. They were microwaved for 45 sec in a "cool" spot located by using the neon bulb display method with the temperature probe in the fixative and temperature feedback limit set to 37C.
Processing for Plastic-embedded Tissue in the Microwave
After washing at room temperature (RT), liver blocks were dehydrated in the MW oven through a graded series of aqueous ethanol solutions from 50 to 100% twice for 45 sec for each step at 45C, infiltrated with 1:1 ethanol:resin (low-viscosity Spurr's resin, Unicryl, or LR White) for 15 min, temperature set to 50C, then 100% of resin three times for 10 min at 50C with a restriction of 5 min. The tissue was transferred into BEEM capsules filled with fresh resin and polymerized as described by
Labeling of Plastic-embedded Sections in the Microwave
Immunolabeling was carried out at 37C unless otherwise specified. Briefly, the grids were floated on 30 µl of PBS containing 0.1% Tween-20 (PBST), followed by PBST containing 1% bovine serum albumin, 0.1% coldwater fish skin gelatin (Sigma Chemical; St Louis, MO) (PBST + BG), and irradiated for 5 min for each step at 37C to block nonspecific labeling with the MW oven probe in a "dummy drop" of solution. The grids were then transferred to the primary rabbit anti-catalase (Chemicon International; Temecula, CA) used at 10 µg/ml in PBST+BG and microwaved at 37C for 5, 10, or 15 min with or without a 2-min break in between irradiations. The grids were washed at RT with PBST+BG and incubated with 10-nm gold adducts of goat anti-rabbit IgG (BioCell; Cardiff, UK), used at 1:50 in blocking solution, in the same manner as the primary antibody. The grids were then washed in PBS and distilled water at RT, dried, and stained with 1% ethanolic uranyl acetate for 30 sec at 37C, then washed in distilled water and dried. Using MWI, the processing time from fixation to polymerization was substantially shortened (Table 1).
|
Immunolabeling and Thin Plastic Embedding of Frozen Sections in the Microwave
Cryoultramicrotomy was carried out essentially according to
The immunolabeled frozen sections were then processed for ultrathin plastic embedding as previously described, except that the processing was done in the MW oven instead of at RT (
|
Determination of Immunolabeling Density
Immunolabeled sections were viewed in a Philips CM12 TEM. Gold particles were counted in 10 randomly chosen peroxisomes in the hepatocytes. Ten micrographs of peroxisomes from liver tissue processed by each method were captured with a GATAN Retractable Multiscan digital camera. The number of gold particles in a 0.2-µm2 area of peroxisomes was determined with Digital Micrograph software and was used to extrapolate the average number of particles per µm2.
![]() |
Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since the pioneering work of
Representative micrographs of sections from LR White-embedded rat liver fixed, processed, and immunolabeled in the MW oven with a polyclonal anti-catalase antibody are shown in Fig 1. Frozen sections similarly labeled and embedded in the MW by the ultrathin plastic embedding method are shown in Fig 2. The liver samples for both LR White and frozen sections were fixed by MWI in a primary fixative consisting of 3% formaldehyde and 0.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, which, in our experience, usually results in satisfactory preservation of cell structure and retention of immunoreactivity. The procedure we recommend for MW primary fixation and the subsequent immunolabeling of LR White or frozen sections is 45 sec of irradiation in the fixative solution with a limit of 37C, although shorter irradiation times on the order of 6 sec were reported to result in good preservation of the ultrastructure upon further processing in osmium and plastic embedding (
|
|
Although good preservation of ultrastructure can be obtained in the absence of osmium, the organelle membranes are usually not contrasted in plastic-embedded samples that have not been osmicated. In the past, although cryoultramicrotomy provided high immunoreactivity because of osmium treatment, dehydration, infiltration, and resin embedding omission, the ultrastructural preservation of frozen sections was often unsatisfactory. However, when the method of ultrathin embedding is carried out either at RT or in the MW as described here, frozen sections that are postosmicated after the immunolabeling step retain the detailed ultrastructural delineation of conventional osmium staining and plastic embedding (
Immunolabeling Density in Sections of Liver Embedded in Acrylic Resins
We found that the labeling density for peroxisomal catalase in sections from liver embedded in Spurr's resin was low, even after etching with metaperiodate. Unicryl-embedded liver samples prepared by MWI suffered from poor morphology, although the immunolabeling density was comparable to that obtained on LR White-embedded liver sections. Consequently, we did not pursue experimenting with these resins.
We did find that the immunolabeling density of LR White sections from blocks fixed and processed by MWI was substantially increased over that the samples labeled the conventional way. Whereas the immunolabeling density was 118 ± 26 particles/µm2 for the samples labeled at RT, continuous irradiation for 5, 10, or 15 min resulted in an immunolabeling density of 90 ± 10, 273 ± 24, and 448 ± 25 particles/µm2, respectively. When a 2-min break after every 5 min of MWI was allowed, the labeling density increased to 111 ± 12, 290 ± 28, and 447 ± 48 particles/µm2 after 5, 10, and 15 min, respectively (Fig 3).
|
Immunolabeling Density on Liver Frozen Sections
We made similar observations when we quantified the number of gold particles obtained on sections fixed and immunolabeled by the conventional method of preparation vs MWI. When frozen sections were incubated at RT for 30 min each in the primary and secondary antibodies, the labeling density was 146 ± 13 gold particles per µm2 of peroxisomal matrix. The samples irradiated continuously displayed a low labeling density at 5 min (51 ± 5 gold particles per µm2), which increases incrementally at 10 min (203 ± 20) and 15 min (287 ± 24). The labeling density increased even more dramatically when a 2-min pause was allowed in between irradiations. At 5-min irradiation with a pause, the labeling density was 140 ± 16 particles/µm2, approximately the same density achieved with conventional techniques (146 ± 13). At 10 min, the labeling density increased to 301 ± 38 particles/µm2, and at 15 minutes to 472 ± 48 particles/µm2, an almost fourfold gain over the conventional immunolabeling protocol. The longer periods of irradiation, along with a 2-min pause after every 5 min, showed a labeling density that was higher than that achieved with the conventional method and with continuous irradiation (Fig 3). In both plastic-embedded and frozen sections, the nonspecific background level remained low and was not enhanced by MWI.
After 15 min of irradiation, the increase in labeling density plateaued in both plastic-embedded and frozen sections (data not shown). The plateau may be caused by detrimental effects of evaporation causing changes in the concentration of the antibody, molarity of the salts, and strength of the pH (
To summarize, MWI provides an effective method for high-resolution immunoelectron microscopy that can be applied to the processing and immunolabeling not only of plastic-embedded but also of frozen sections. Because the immunolabeling density of samples in LR White (448 ± 25 particles/µm2) and frozen sections (472 ± 48 particles/µm2) was fairly similar after 15 min of MWI (Fig 3), we recommend carrying out immunolabeling experiments in LR White-embedded material when there are a large number of specimens to be immunolabeled. However, immunolabeling of frozen sections is still our method of choice, especially when the maximal retention of both cellular ultrastructure and antibody binding capacity of protein antigen in the specimen is required.
Received for publication March 6, 2000; accepted March 8, 2000.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Boon ME, Hendrikse FCJ, Kok PG, Bolhuis P, Kok LK (1990) A practical approach to routine immunostaining of paraffin sections in the microwave oven. Histochem J 22:347-352[Medline]
Chicoine L, Webster P (1998) Effect of microwave irradiation on antibody labeling efficiency when applied to ultrathin cryosections through fixed biological material. Microsc Res Tech 42:24-32[Medline]
Choi TS, Whittlesey M, Slap S, Anderson V (1995) Advances in temperature control of microwave immunohistochemistry. Cell Vis 2:151-164
Cuevas EC, Bateman AC, Wilkins BS, Johnson PA, Williams JH, Lee AH, Jones DB, Wright DH (1995) Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies. J Clin Pathol 47:448-452[Abstract]
Giammara B (1993) Microwave embedment for light and electron microscopy using epoxy resins, LR White, and other polymers. Scanning 15:82-87
Giberson RT, Demaree RS, Jr, Nordhausen RW (1997) Four-hour processing of clinical/diagnostics specimens for electron microscopy using microwave technique. J Vet Diagn Invest 9:61-67[Medline]
Giberson RT, Smith RL, Demaree RS (1995) Microwave fixation: understanding the variables to achieve rapid reproducible results. Microsc Res Tech 32:246-254[Medline]
Gu J (1994) Microwave Immunocytochemistry. In Gu J, Hacker GW, eds. Modern Methods in Analytical Morphology. New York, Plenum Press, 67-80
Gu J, Choi T-S, Whittlesey M, Slap S, Anderson V (1995) Development of microwave immunohistochemistry. Cell Vis 2:257-259
Hjerpe A, Boon ME, Kok LP (1988) Microwave stimulation of an immunological reaction (CEA/anti-CEA) and its use in immunohistochemistry. Histochem J 20:388-396[Medline]
Keller GA, Krisans SK, Maher P, Scallen TJ, Singer SJ (1989) Sub-cellular localization of sterol-carrier protein 2 in rat hepatocytes: its primary localization to peroxisomes. J Cell Biol 108:1353-1361[Abstract]
Keller GA, Tokuyasu KT, Dutton AH, Singer SJ (1984) An improved procedure for immunoelectron microscopy: ultrathin plastic embedding of immunolabeled ultrathin frozen sections. Proc Natl Acad Sci USA 81:5744-5747[Abstract]
Kok LP, Boon ME, Smid HM (1993) The problem of hot spots in microwave used for preparatory techniquestheory and practice. Scanning 15:100-109
Leong AS, Daymmon ME, Milios J (1985) Microwave irradiation as a form of fixation for light and electron microscopy. J Pathol 146:313-321[Medline]
Login GR, Dvorak AM (1988) Microwave fixation provides excellent preservation of tissue, cells and antigens for light and electron microscopy. Histochem J 20:373-387[Medline]
Login GR, Dvorak AM (1993) A review of rapid microwave fixation technology: its expanding niche in morphological studies. Scanning 15:58-66[Medline]
Login GR, Dvorak AM (1994) Methods of microwave fixation for microscopy. A review of research and clinical applications: 1970-1992. Prog Histochem Cytochem 27:1-27[Medline]
Login GR, Galli SJ, Dvorak AM (1992) Immunocytochemical localization of histamine in secretary granules of rat peritoneal mast cells with conventional or rapid microwave fixation and an ultrastructural post-embedding immunogold technique. J Histochem Cytochem 40:1247-1256
Login GR, Galli SJ, Morgan E, Arizono N, Schwartz LB, Dvorak AM (1987) Rapid microwave fixation of rat mast cells. I. Localization of granule chymase with an ultrastructural postembedding immunogold technique. Lab Invest 57:592-599[Medline]
Login GR, Ku TC, Dvorak AM (1995) Rapid primary microwavealdehyde and microwaveosmium fixation improved detection of the parotid acinar cell secretory granule alpha-amylase using a post-embedding immunogold ultrastructural morphometric analysis. J Histochem Cytochem 43:515-523
Mayers CP (1970) Histological fixation by microwave heating. J Clin Pathol 23:273-275[Medline]
Naganuma H, Ohtani H, Nagura H (1990) Immunoelectron microscopic localization of aromatase in human placenta and ovary using microwave fixation. J Histochem Cytochem 38:1427-1432[Abstract]
Tokuyasu KT (1980) Immunochemistry on ultrathin frozen sections. Histochem J 12:381-403[Medline]
van den Bosch H, Schutgens RB, Wanders RJ, Tager JM (1992) Biochemistry of peroxisomes. Ann Rev Biochem 61:157-197[Medline]
Werner M, Von Wasielewski R, Komminoth P (1996) Antigen retrieval, signal amplification and intensification in immunohistochemistry. Histochem Cell Biol 105:253-260[Medline]
Zondervan PE, De Jong A, Sorber CW, Kok LP, De Bruijn WC, Van der Kwast TH (1988) Microwavestimulated incubation in immunoelectron microscopy: a quantitative study. Histochem J 20:359-364[Medline]