Amersham AuroProbe. GE Healthcare. Product Booklet

Transcription

1 GE Healthcare Amersham AuroProbe Secondary antibody coated colloidal gold probes for use in the electron microscopical immunogold (silver) staining technique Product Booklet Codes: RPN420 RPN421 RPN422 RPN424 RPN425 RPN430 RPN431 RPN444

2 Page finder 1. Legal 3 2. Handling Safety warnings and precautions Quality control and stability 4 3. Product information 6 4. Procedures for immunoelectron microscopy Surface immunomarking of cell suspensions On-grid immunomarking Pre-embedding immunomarking of intracellular antigens in cultured cell monolayers Silver enhancement of colloidal gold probes References Ordering information 26 2

3 1. Legal GE and GE monogram are trademarks of General Electric Company. Amersham, AuroProbe and IntenSE are trademarks of GE Healthcare companies. AuroDye and IntenSE are under licence of Jassen Biotech NV, B2430, Olen, Belgium. GE Healthcare reserves the right, subject to any regulatory and contractual approval if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your GE Representative for the most current information and a copy of the terms and conditions 2006 General Electric Company All rights reserved. GE Healthcare UK Limited. Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK 3

4 2. Handling 2.1. Safety warnings and precautions Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice. Warning: Contains sodium azide in dilute solution. Dispose of waste by flushing with copious amounts of water to avoid the build up of explosive metallic azides in copper and lead plumbing. The total azide present in each pack is 525 µg. Note that the protocol may require the use of sodium azide, glutaraldehyde, sodium borohydride, osmium tetroxide, cacodylic acid, paraformaldehyde, methanol, ethanol and other laboratory chemicals. Please follow the manufacturers safety data sheets for the safe handling and use of these materials Quality control and stability The activity of each batch is measured at regular intervals using a quantitative test, based on the procedure of Moeremans et al (J. Immunol. Meth., 74, p.353, 1984). In this test, 0.5 ml of a 1:25 dilution 4

5 of the probe is reacted, at room temperature for 2 hours, with a dilution series of the corresponding antigen (25 ng 0.1 ng/spot) spotted onto a nitrocellulose strip. The last visible spot indicates the activity of the batch, from which any deviations as a function of time can be monitored. Results of such tests have provided evidence for the stability of the AuroProbe EM reagents. Reagents are also inspected for the presence of aggregates; more than 75% of the gold particles must be singlets, and less than 5% should occur in aggregates of 3 or more particles. AuroProbe EM reagents have a guaranteed shelf-life of one year from the date of analysis. 5

6 3. Product information Three separate specificities of secondary antibodies, all affinitypurified on the corresponding antigen, are available. The anti-mouse IgG and IgM antibodies are cross-adsorbed to eliminate crossreaction. A mixed anti-mouse IgG + IgM specificity is also available, which recognizes both IgM and IgG. Anti-rabbit IgG and anti-mouse IgG are available, coupled to 5, 10 and 15 nm gold particles. Anti-mouse IgG+IgM is available, coupled to 5 and 10 nm gold particles. The different sizes are not overlapping. The AuroProbe EM* reagents are manufactured and purified according to a novel large-scale production method. This method eliminates virtually all free antibodies and aggregates of gold particles and generates very narrow and constant size distributions. The particle size distribution and batch number are given on a separate sheet. Information on the relative paucity of clumps is also provided. The absence of clumps is essential for a high quality gold probe. AuroProbe EM reagents are calibrated to have an OD at 520 nm of 2.5 for the 5 and 10 nm sizes, 3.5 for the 15 nm and 7.0 for the 30 nm sizes. They are suspended in 20 mm Tris buffered saline, ph 8.2, supplemented with 0.1% BSA and 0.05% sodium azide: 0.1% BSA-Tris. Store at 2 8 C. Do not freeze. * Patents: USA / / and other related patents worldwide. 6

7 4. Procedures for immunoelectron microscopy 4.1. Surface immunomarking of cell suspensions Preparation of cells and fixation Suspensions at a minimum of 10 7 cells/ml are used. 100 µl is used per specimen. The cells can be used alive or be prefixed. Live cells are, in general, kept on ice or in the presence of 0.2% sodium azide to prevent internalization of the antibodies. The type of fixative depends on the resistance of the antigenic determinants to fixation. After fixation, the cells are washed several times. When glutaraldehyde concentrations higher than 0.3% are used, it may be advantageous to treat the cells with NaBH 4, 1 mg/ml in fixation buffer, for 2 x 5 minutes. NaBH 4 should be stored in a desiccator and dissolved just prior to use. After the final wash, the cells are transferred to the buffer used for the antibody incubations. Immunomarking Phosphate buffered saline (PBS), calcium and magnesium free, 0.01 M phosphate, containing 0.15 M sodium chloride, ph , supplemented with 1% BSA, is used as buffer for antibody incubations and washing: wash buffer. For living human blood cells, the addition of 1% heat inactivated Ab-serum helps in preventing Fc binding by Fc receptors. When working at room temperature, 0.2% sodium azide is also added. 1. Pipette 100 µl resuspended cells at 10 7 cells/ml into test tubes with a conical tip. 2. Add 5 µl appropriately reconstituted or diluted antibody or antiserum to each test tube. 7

8 3. Mix well and incubate for 30 minutes or longer. Agitate at least every ten minutes. When azide is used, limit the incubation time to 30 minutes. 4. Add 2 ml wash buffer to the tubes and centrifuge at 300 xg for five minutes. 5. Withdraw the wash buffer completely with a Pasteur pipette linked to a vacuum system. Do not disturb the cell pellet. 6. Repeat steps 4 and 5. Resuspend the cells before centrifuging. 7. Add 25 µl of an appropriately diluted AuroProbe EM reagent. The dilution is made in wash buffer, or optionally 0.1 to 1% BSA-Tris. BSA-Tris is a buffer composed of 20 mm Tris buffered saline, ph 8.2, supplemented with BSA, in the w/v percentage indicated by the preceding figure. For prolonged incubations, 20 mm sodium azide may be added. The optimum dilution must be experimentally determined. The recommended range is from undiluted to 1/ Mix well with the cells and incubate for 30 minutes or longer. Agitate at least every ten minutes, to avoid clumping of labelled cells. 9. Wash as in steps 4, 5 and Add 2 ml 2% glutaraldehyde in 0.1% cacodylate buffer, ph 7.2 for electron microscopy. Incubate at room temperature for 30 minutes. 11. Wash 3 times with fixation buffer. 12. Treat with 2% OsO 4 ** in the cacodylate buffer at room temperature for 20 minutes. 13. Wash in water and stain en bloc with uranyl acetate. 14. Embed in a plastic resin. ** Warning: OsO 4 should be handled in a fume hood. Gloves and safety glasses should be worn. 8

9 4.2. On-grid immunomarking Immunomarking of ultra-thin sections of resin-embedded tissues or cells The fixation method and embedding material used will depend on the kind of antigen. It is recommended that a range of fixatives are tried for every antigen. Usually postfixation with OsO 4 is omitted but there are examples of successful marking of OsO 4 fixed material. Various types of resin have been used, with varying success. Hydrophilic resins such as the Lowicryls, LR White and glycol methacrylate, in general, yield the best immunoreactivities. Lowicryl K4M may be combined with low temperature dehydration and polymerization at -35 C. This produces good ultrastructural preservation (1) of tissues not treated with OsO 4. A combination of fixation with buffered glutaraldehydepicric acid and embedding at room temperature in LR White from 7% ethanol has also given satisfactory preservation (2). LR White embedding has been used with good results in combination with conventional glutaraldehyde/oso 4 fixation of plant tissues (3). Tissue sections embedded in hydrophobic resins, or fixed with aldehyde and OsO 4 need etching before incubation with immune reagents. These sections are preferably mounted on nickel or gold grids. There are several ways of processing electron microscope grids. Grids may be treated by floating on top of drops of reagent dispensed onto sheets of parafilm. Washing can take place on larger drops of buffer, or by jet washing which entails rinsing individual grids held in forceps, with a stream of buffer from a syringe. If a large series of grids or coated slot grids need to be processed, we recommend the procedure outlined below, for which the following extra materials are needed:- 1. Microtest plates with small wells (for example Falcon 3034, Falcon Plastics) able to firmly hold drops at µl without a risk of 9

10 cross contamination. The lid of the plate also helps to prevent contamination and keeps the air humid. 2. A 48-well culture plate, for the washing steps. 3. A pair of fine pointed forceps, for handling the grids. Alternately a metal loop of 3.2 mm internal diameter made of nickel-coated copper wire of 0.2 mm thickness, and flattened between the flat beaks of a vice, will diminish the risk of contamination, and greatly facilitate transfer of grids. 4. A rocking table, for example the Heidolph Reax III. For incubations with immune reagents, the grids are floated, sections downward on top of drops of immune reagents displayed in wells of the microtest plate. For rinsing or washing, the grids are floated on the meniscus of the washing fluid, dispensed into a well of the 48-well plate. The fluid is kept in constant motion by putting the 48-well plate on the rocking table. The grids move around and this yields a smooth but very efficient type of washing. The grids are transferred with either the fine forceps or preferably with the flattened loop (see above). Procedure Buffers Either 0.1% BSA-Tris (for secondary antibody probes) or PBSG, consisting of PBS supplemented with 0.5% BSA and 0.1% gelatin (E Merck FRG, Art no. 4070) are recommended. The latter is suitable for both secondary antibody and protein A probes. The use of Gelatin with Bloom number has been recommended by Behnke et al (5). 0.1% BSA-Tris is a buffer composed of 20 mm Tris buffered saline supplemented with 1 mg/ml BSA and 20 mm NaN 3. These buffers are used throughout the procedure. The addition of 0.05% Tween 20* and 0.5 M NaCl to both the dilution and washing * Tween 20 is a registered trademark of Atlas Chemical Industries Inc 10

11 buffer may help to reduce background problems (6). All steps are carried out at room temperature. Grids are transferred from drop to drop with sections downwards. 1. The grids are rinsed on drops of water for 10 minutes. 2. If required, the etching step should be carried out here. For an evaluation of various etching procedures, see Bendayan and Zollinger (7). In this paper, a pretreatment of the thin sections with a saturated aqueous solution of sodium metaperiodate for minutes is recommended. According to Craig et al (8) good results are achieved with LR white etched for 10 minutes with saturated sodium metaperiodate, followed by a rinse in water and 10 minutes 0.1 M HCl. In general etching is done by floating the grids on drops of etching solution in the wells of a microtest plate. 3. After etching, grids are rinsed on water for 5 minutes. 4. The washed grids are optionally incubated on drops of buffer supplemented with 5% normal serum for 15 minutes. 5. Grids are then incubated for minutes with drops of specific primary antibody (Ab), preferably affinity purified, at 2 to 25 µg/ml, or with a high dilution of antiserum (>500x) in buffer, supplemented with 1% normal serum (same species as secondary antibody). Antibody concentration and incubation time may be optimized by testing an Ab dilution series with varying incubation times. 6. Grids are transferred to the washing buffer for 2 x 5 minutes. 7. The grids are then incubated for 30 minutes with drops of the AuroProbe EM reagent, diluted just before use with buffer to an OD at 520 nm of 0.1 (approximately 1 in 25). It is recommended that a series of dilutions from 1 in 10 to 1 in 200 is tried with new localization experiments. 11

12 8. The washing in step 6 is repeated, followed by two washes in PBS, and postfixed with 2% glutaraldhyde in PBS. 9. The grids are washed with distilled water (2 x 5 minutes) and contrasted using conventional techniques. For double marking, two primary antibodies (dilutions: see above), produced in two different animal species are mixed and applied simultaneously (step 5). After the washing step, a mixture of the corresponding AuroProbe EM reagents (dilutions: see above) with two non-overlapping sizes is applied (step 7). An alternative and attractive method for double marking of resin sections is Bendayan s two-face technique (9). For this technique, uncoated grids must be used. The first two steps take place by floating the sections on drops, face A downward. After the rinsing of step 8, the whole procedure is repeated with the second two steps, face B downward. A possible pitfall of this two-face technique may result in false negative results: when a structure, for example a storage granule, is small or cut tangentially, its contents may be exposed only on one side of the section. Immunomarking of ultrathin cryosections (Modified from Geuze et al (10) and Slot and Geuze (11).) Ultrathin cryosections are prepared from glutaraldehyde and/or paraformaldehyde fixed tissue after infusion with 2.3 M sucrose according to Tokuyasu (12). They are picked up on carbon-coated nickel or gold grids. For incubation with immune reagents they are floated sections downward on top of drops of solutions dispensed into wells of a micro-test plate. All steps are carried out at room temperature. For rinsing or washing, the grids are floated on the meniscus of a rinsing or washing fluid put into a well of a 48-well plate (see the description above) with either the fine forceps or preferably with the flattened 12

13 loop (see page 10). The plate should be agitated throughout by means of a rocking table. Buffers: as buffer systems either 0.1% BSA-Tris (for secondary antibody probes) or PBSG, consisting of PBS supplemented with 0.5% BSA and 0.1% gelatin (E. Merck, FRG Art No 4070) are recommended. The latter is suitable for both secondary antibody and protein A probes. The use of gelatin with Bloom number has been recommended by Behnke et al (13). 1. Grids are incubated on 0.02 M glycine in buffer for 10 minutes. Alternatively one can use 1% sodium borohydride (freshly prepared from dry NaBH 4 (stored in a desiccator)) in PBS for 10 minutes. 2. Rinse with buffer. 2 x 1 minute. 3. Rinsed grids are placed on drops of buffer supplemented with 5% normal serum (same species as secondary antibody) for 15 minutes. 4. Incubation with specific primary antibody for 30 to 60 minutes. Antibody should preferably be affinity purified, 2 20 µg/ml, or a high dilution of high titre antiserum in buffer supplemented with 1% normal serum (same species as secondary antibody). Antibody concentration and incubation time may be optimized by testing an Ab dilution series with a variety of incubation times. 5. Grids are rinsed with buffer 2 x 5 minutes. 6. Incubation with appropriate Auroprobe EM reagent for 30 minutes to two hours. The probe should be diluted, just before use, with buffer, to an OD at 520 nm of approximately 0.1 (approximately 1 in 25). It is recommended that a series of dilutions from 1 in 10 to 1 in 200 is tried with new localization experiments. 7. Grids are rinsed with buffer 2 x 5 minutes; drops of approximately 250 µl or larger. 13

14 8. Grids are washed twice with PBS and postfixed in 2% glutaraldehyde in PBS. For double marking, two primary antibodies (dilution: see page 13), produced in two different animal species are mixed and applied simultaneously (step 4). After the washing step, a mixture of the corresponding AuroProbe EM reagents (dilution: see page 13) with two non-overlapping sizes is applied (step 6). The sections can be uranyl-stained and embedded in methylcellulose after Tokuyasu as modified by Griffiths et al (14), by passing them on top of approx 250 µl drops (dispensed on a parafilm sheet) of the following solutions at room temperature. 1. Distilled water, 3 x a few seconds. 2. 2% uranyl acetate in 0.15 M oxalic acid, brought to ph 7 with 5% NH 4 OH, 10 minutes. 3. Distilled water, 3 x a few seconds to 4% uranyl acetate in distilled water approx 10 minutes. 5. Distilled water, approximately 1 second. Care should be taken as uranyl acetate is easily extracted. This step can be replaced by 2 or 3 fast passages with drops of 1.5% methylcellulose % methylcellulose solution is prepared by adding 1.5 g methylcellulose (Tylose MH300 or Methocel MC400CP, Fluka) to 100 ml distilled water. The solution is stirred at 2 8 C for 24 hours or more, and stored in a refrigerator. The methylcellulose is centrifuged in an Eppendorf type tube at approximately g for 5 minutes just before use. 7. The grids must be picked up from the methylcellulose drop within 30 seconds with the flattened wire loop (see page 10). Excess methylcellulose is removed with filter paper, leaving the hole of the loop evenly filled. The grid is allowed to dry before removing it from the loop. 14

15 The above procedure produces good delineation of membranous structures, but cytoplasmic filaments and other structures are often not visible. Plastic embedding and subsequent contrasting of the section gives an image closely approaching that of a conventional thin section. The basic procedure is as follows, starting from step 7 of the immunostaining procedure. It can be adapted to the special requirements of different specimens (15). The grids are immersed in subsequent solutions for postfixation, staining, dehydration and embedding. All steps are carried out at room temperature. 1. Wash the grids in H 2 O or 0.05 M cacodylate buffer, ph 7.4 for 3 x 2 minutes. 2. Postfix with 2% OsO 4 in 0.05 M cacodylate buffer, ph 7.4 for 10 to 20 minutes. 3. Wash for 3 x 2 minutes with H 2 O or 0.05 M cacodylate buffer, ph Stain with uranyl acetate in veronal buffer, ph 6.4, for 10 to 20 minutes. For 100 ml veronal buffer: 20 ml veronal acetate stock solution (0.28 M), 28 ml 0.1 M HCl, add water to 100 ml, add uranyl acetate (crystalline) to 0.05% w/v. 5. Wash in H 2 O for 3 x 2 minutes. 6. Dehydrate through 40%, 75%, 95% and 2 x 100% ethanol for 2 minutes each. 7. Leave in 50/50 ethanol/lr White for 10 minutes. 8. Leave in 100% LR White for 2 x 10 minutes. 15

16 9. Place the grids between 2 #50 hardened Whatman filter papers and apply slight pressure to remove liquid. 10. Place them in the oven at 60 C for 1 to 2 hours. 11. The sections can now be countersigned using conventional uranyl acetate and lead citrate staining, or alternatively with the Locke and Huie bismuth stain (16) Pre-embedding immunomarking of intracellular antigens in cultured cell monolayers. Introduction Immunocytochemical methods are widely used to investigate the relative position of structural and regulatory proteins in cell cultured monolayers. Light microscope techniques are well established, especially for cytoskeletal proteins, but the EM - localization of these antigens has been far less successful. The diameter of the molecules and the subcellular structures are often smaller than the thickness of the section, and not precisely orientated and thus may not be exposed to the probe. Additionally, as the probe cannot penetrate the resin, post embedding immunomarking produces only limited information. Ultra-thin frozen sections which would provide the correct tissue orientation required to correlate the staining pattern with specific subcellular structures and with the light microscopic data from whole cells, cannot yet be obtained. In addition, it is still uncertain whether this technique can produce the necessary ultrastructural contrast and delineation. In the mean time, the pre-embedding approach remains the method of choice. The cells are pre-fixed, made permeable to the probes and then further processed for electron microscopic observation. A major drawback of the pre-embedding approach is that it tends to damage the membrane systems, so that it cannot be used to study, 16

17 for example, cytoskeleton-membrane interactions. More gentle permeabilization techniques using saponin or digitonin in general do not render the cells permeable enough to gold probes. In many instances, it is necessary to extract the cells to variable degrees with a detergent in a suitable buffer before fixation. Glutaraldehyde is practically the only fixative that gives sufficient preservation of fine structural details. In some instances, however, fixatives such as paraformaldehyde or even cold methanol or ethanol, followed by aldehyde fixation before and/or after the immunolabelling procedure, can yield the necessary information (for example, see (17)). The smallest gold probes will obviously penetrate better and give the best spatial resolution. In cultures, the quantity of antigen to be detected with gold probes can be fairly high as compared with that in a thin section. Therefore, it is often necessary to use our gold probes at low dilutions or even undiluted. We have also found that the marking of cells may not be complete at all sites, especially in places where the filaments are arranged in dense arrays. Specially adapted postfixation and contrasting methods make the correlation of the probe with recognizable structures easier. As a whole, the researcher will have to consider the kind of information he wants to obtain, and work out conditions that can produce meaningful results. Often, electron microscopical work is done in conjunction with light microscopical techniques. Our AuroProbe LM reagents for Immunogold Silver Staining (IGSS) allow visualization of the distribution of the gold probes at the light microscopical level, and the comparison of this with the results obtained, by, say, indirect immunofluorescence (18). 17

18 This introduction is intended to assist you in obtaining the best possible results. The following recommended procedures have led to good results (19,20,21). Our products have been manufactured with great care, but in this field it is not possible to predict whether good results will be obtained. The quality and avidity of the primary antibody are of crucial importance. In the majority of cases polyclonal antibodies need to be purified by affinity chromatography. Cell culture Cells may be grown under standard conditions on round or 18 x 18 mm coverslips in plastic petri-dishes, or directly in plastic petri-dishes. Treating the surface of the coverslip with a release agent, for example MS-123 release agent, dry lubricant (Miller- Stephenson Chemical Company, Inc, Danbury, CT 06810, USA), will help separate the glass coverslip from the epoxy resin after flat embedding. The release agent is sprayed on the cell-side of the coverslip and immediately wiped dry with a paper tissue. Caution: avoid skin contact with and breathing vapour of the release agent. Consult the manufacturer s safety instructions. For cells grown on petri-dishes, Permanox dishes (Lux, Lab-Tek Division, Miles Laboratories Inc, Naperville, IL 60540, USA) are recommended, because they are resistant to acetone and epoxy resins. It is recommended that the cells are used when they have reached about 50% confluency. Handling: When the cells are grown on larger coverslips or on plastic, most of the steps, for example fixation, permeabilization, incubation with immune reagents, washing, are done in the petridish in which the cells were grown. Removal of the liquids may be done with a Pasteur pipette, fixed in a stand and connected to a vacuum flask. This set-up greatly facilitates the handling. 18

19 Fixation-permeabilization Use of glutaraldehyde-fixation The glutaraldehyde needs to be of high purity, free from polymers. Glutaraldehyde can be used directly on living cells at 0.3 to 1% or mixed with variable concentrations of a detergent, for example Triton X-100* at 0.1% to 1%, for about 10 minutes. Glutaraldehyde fixation can be preceded by extraction with a detergent, such as Triton X-100 at 0.1 to 0.5% for 20 seconds or longer. Any suitable buffer system can be used. Good results for studying the microfilament system (Small and Celis (1978)) have been obtained using the following buffer: HBBS (NaCl, 137 mm; KCl, 5 nm; Na 2 HPO 4, 1.1 mm; KH 2 PO 4, 0.4 mm; NaHCO 3, 4 mm; glucose, 5.5 mm); PIPES, 5 mm; MgCl 2, 2 mm; EGTA, 2 mm. Adjust the ph to 6.1 with 1 N NaOH. If cells are extracted with a detergent before fixation, the EGTA is left out. Another buffer useful when the microtubular system is investigated is: 65 mm PIPES, 25 mm HEPES, 10 mm EGTA and 2 mm MgCl 2, ph 6.9 (22). Fixation and/or extraction are done in the petri-dish after washing the cells with PBS without Mg 2+ and Ca 2+. The washing fluid is removed and fixation fluid, optionally preceded by extraction fluid, is added with a Pasteur pipette. Once the cells are fixed, they are rinsed with several changes of fixation buffer and washed for 3 x 10 minutes in PBS without Mg 2+ and Ca 2+. The cells are permeabilized for 30 minutes in 0.2 to 0.5% Triton X-100 in fixation buffer. Do not include this step when surface labelling is the purpose. Other permeabilization methods exist and can be tried. This step is followed by a treatment with NaBH 4, 1 mg/ml in fixation buffer, twice for 10 minutes. The NaBH 4 is very hygroscopic and should be stored in a desiccator, and only be dissolved just prior to use. The cells are washed with fixation buffer, and then left in 0.1% BSA-Tris for immunocytochemistry. * Triton X-100 is a registered trademark of Rohm and Haas Co. 19

20 Use of paraformaldehyde. The same procedure can be used with 2 to 4% freshly prepared paraformaldehyde, optionally substituted with 0.05 to 0.1% glutaraldehyde. The permeabilization step can be done with lower concentrations of detergent and the NaBH 4 can be omitted. Use of cold methanol and ethanol The cells are washed with PBS and fixed for 10 minutes in cold (-20 C) absolute methanol or ethanol. The fixative is replaced after a few seconds. The cells are washed 3 x 5 minutes with PBS and then transferred to the 0.1% BSA-Tris. Other buffers may also be used. Incubation with immune reagents When the cells are grown on coverslips, the fluid is carefully removed from around the coverslip and, depending on the size of the coverslip, 30 to 70 µl incubation fluid is pipetted from an adjustable micropipette. When the cells are grown on plastic, a circle with a diameter of approximately 2 cm is drawn with a sharp object around the cap of, for example, a culture medium flask, held in place over the centre of the petri-dish bottom. Only cells within this circle are used. Cells outside the circle are excluded by wiping the area dry with paper, and pipetting the incubation fluid (approx 100 µl) within the encircled area. Incubate the cells with the following reagents: 1. 5% normal serum (NS) in 0.1% BSA-Tris for 20 minutes. 2. The primary antibody at 1 to 10 µg/ml in 1% BSA-Tris for 1 to 12 hours. 3. Wash with 0.1% BSA-Tris, 3 x 10 minutes. 4. An appropriate AuroProbe EM reagent, diluted in 0.1% BSA-Tris. 5. Wash as in step Postfixation and embedding procedure. 20

21 (NS) means heat inactivated normal serum, of the same species as the secondary antibody). Postfixation and embedding procedure The cells can be postfixed with glutaraldehyde and osmium tetroxide and processed for embedding using standard methods. In some instances, especially for microfilaments, the following procedure produces good preservation and contrast (23). The cells from step 5 are further washed twice in Sörensen s phosphate buffer, ph 7.2 (Phosphate inorganic buffer: 36 ml 0.2 M Na 2 HPO ml 0.2 M NaH 2 PO 4 diluted to 100 ml with H 2 O). They are postfixed in 1% glutaraldehyde + 0.2% tannic acid in Pi buffer. They are then further postfixed in 0.5% osmium tetroxide in Pi buffer, 10 minutes on ice and washed in Pi buffer for 3 x 5 minutes. The cells are partially dehydrated in 70% ethanol (several changes) impregnated with 0.5% uranyl acetate + 1% phosphotungstic acid in 70% ethanol for 30 minutes, further dehydrated in alcohol and embedded in epon. Cells grown on coverslips are flat embedded on microscope slides treated with the same release agent as used for coverslips. Spacers are formed by pieces of coverslips or any other appropriate material. A drop of epon is put on the slide and the coverslip, cells downward, is positioned on the spacers. The epon is hardened for 3 days at 50 C and is separated from the glass slide and coverslip by alternately dipping in boiling water and liquid nitrogen. Suitable cells are selected and circled with a diamond. They are cut out of the epon and re-embedded, for either horizontal sectioning (cell side up) or for vertical sectioning (cells vertically). 21

22 4.4. Silver enhancement of colloidal gold probes Introduction For applications in scanning electron microscopy, the limited resolution power of scanning electron microscopes necessitates the use of particles of at least 15 nm. This results in low density labelling, as the density is inversely related to the probe size (24). The detection efficiency is higher when small gold particles (for example 5 nm) are employed. For applications in transmission electron microscopy, for which small gold particles (for example 5 nm) are used, relatively high magnifications are required for visualization of the marker. This makes it impossible with some applications to obtain an overall picture of the antigenic distribution within a cell. By applying a silver enhancement procedure (25 29) the highest efficiency of labelling using 5nm probes is fully exploited. Holgate et al (30) and Danscher (31) have shown that metallic silver will precipitate around colloidal gold particles. During the development phase, gold particles are encapsulated in growing shells of silver, resulting in an increase in the size of the gold marker, thus increasing its visibility/detectability. IntenSE M* (RPN 491), from GE Healthcare is an easy-to-use kit for this purpose, developed from a novel and patented chemistry. Procedure General information on silver enhancement is outlined in the package insert provided with each IntenSE M kit. Please read this information carefully. Sections mounted on copper grids cannot be used for silver enhancement. For this application nickel grids are necessary. 1. After the incubation step with AuroProbe EM, wash the specimen and postfix with 2% glutaraldehyde in PBS as described on page 19. * patents pending 22

23 2. Wash the specimen 3 x 5 minutes in PBS and 3 x 3 minutes in distilled water with agitation. 3. Prepare the silver enhancement reagent by mixing equal drops of reagent A and reagent B in a small tube. Mixing of these solutions should be performed immediately prior to the silver enhancement step, since this mixture is only stable for a limited period. Avoid contact of metallic parts with the silver enhancement mixture. Apply the silver enhancement mixture to the preparation and incubate at constant temperature in order to obtain reproducible results (best at 18 C). The time of silver enhancement should be based upon the amplification that the observer considers adequate. 4. Wash the specimen 2 x 5 minutes in excess distilled water. Attention: OsO 4 ** may remove (part of) the metallic silver deposit. Do not fix with OsO 4 after silver enhancement. ** Warning: OsO 4 should be handled in a fume hood. Gloves and safety glasses should be worn. 23

24 5. References 1. ROTH et al, J.Histochem. Cytochem., 29, p.663, NEWMAN et al, Histochem. J., 15, p.543, CRAIG et al, Cell Biol. Int. Rep., 8, p.879, LEUNISSEN, Immunogold Labelling Methods, CRC Press Inc., Boca Raton FI 33431, in press. 5. BEHNKE et al, Eur.J. Cell Biol., 41, p.326, CRAIG, S. et al, Eur. J. Cell Biol., 28, p.251, BENDAYAN et al, J. Histochem. Cytochem., 31, p.101, CRAIG et al, Cell Biol. Int. Rep., 8, p.879, BENDAYAN et al, J. Histochem. Cytochem., 30, p.81, GEUZE et al, J. Cell Biol., 89, p.653, SLOT et al, Immunochemistry, ed. C. Cuello, p. 323, J. Wiley and Sons publ., Chichester, TOKUYASU, J. Histochem. Cytochem., 31, p.164, BEHNKE et al, Eur. J. Cell Biol., 41, p.326, GRIFFITH et al, J. Cell Biol., 95, p.781, KELLER, G. et al, Proc. Natl. Acad. Sci. USA, 81, p.5744, LOCKE et al, Tissue and Cell, 9, p.347, 1977 and 10, p.365, GEUENS et al, Cell Biol. Int. Rep., 7, p.35, DE MEY et al, In Immunocytochemistry. Practical Applications in Pathology and Biology (Ed. Polak, J., Van Noorden, S.) 2nd Edition. Wright, Bristol, London, DE MEY et al, Cell Biol. Int. Rep., 5, p.889, LANGANGER et al, J. Cell Biol., 99, p.1324, LANGANGER et al, J. Cell Biol., 102, p.200,

25 22. SCHLIWA et al, Proc. Natl. Acad. Sci. USA, 78, p.4329, LANGANGER et al, J. Cell Biol., 99, p.1324, LACKIE et al, Histochem., 83, p.545, BASTHOLM et al, J. Electr. Micr. Techn., 4, p.175, BIENZ et al, J. Histochem. Cytochem., 34, p.1337, BIRRELL et al, J. Electr. Micr. Techn., 5, p.219, GOODE et al, J. Electr. Micr. Techn., 5, p.263, SCOPSI et al, J. Histochem., 86, p.35, HOLGATE et al, J. Histochem. Cytochem., 31, p.938, DANSCHER, G., Histochemistry, 71, p.81,

26 6. Ordering information AuroProbe EM Immunogold reagents Code Product name Biological agent RPN420 AuroProbe EM GAR G5 Goat RPN421 AuroProbe EM GAR G10 anti-rabbit IgG(H+L) RPN422 AuroProbe EM GAR G15 RPN424 AuroProbe EM GAMIgG G5 Goat RPN425 AuroProbe EM GAMIgG G10 anti-mouse IgG(Fc) RPN444 AuroProbe EM GAMIgG G15 RPN430 AuroProbe EM GAMIgG+IgM G5 Goat anti-mouse RPN431 AuroProbe EM GAMIgG+IgM G10 IgG+IgM(H+L) RPN438 AuroProbe EM protein A G10 Protein A RPN442 AuroProbe EM streptavidin G10 Streptavidin AuroProbe EM reagents are available in 1 ml vial IntenSE M silver enhancement system Code Product name Unit RPN491 IntenSE M kit 30 ml Ready to use, light-insensitive silver enhancement reagent at neutral ph, tailored for microscopy. Additional reagents Code Product name Unit PN410 Normal goat serum lyophilized, to be reconstituted in 5 ml RPN412 BSA 25 g 26

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