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    • 专利摘要:
    A method for the detection of metals by autometalography in samples of biological tissue. The present invention relates to a method for the detection of a metal by autometalography, preferably zinc, in a sample of biological tissue, preferably an animal cell culture, characterized in that it comprises carrying out at least one incubation of the sample with a fluorochrome specific to said metal and to the use of this method in any area related to autoometalography, and more specifically in biological studies for medical purposes, for toxicology or related to food contamination. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2697922A1
    申请号:ES201730992
    申请日:2017-07-28
    公开日:2019-01-29
    发明作者:I Marti Xavier Ponsoda；Hinojosa Raúl Ballestin；Garcia Carlos Lopez；Tudela Asunción Molowny
    申请人:Universitat de Valencia；
    IPC主号:
    专利说明:

    [0001]
    [0002] A METHOD FOR THE DETECTION OF METALS BY AUTOMETALOGRAPHY
    [0003]
    [0004] SECTOR OF THE TECHNIQUE
    [0005] The present invention is part of the technology of metal detection methods
    [0006] BACKGROUND OF THE INVENTION
    [0007] The techniques of autometalography are known for decades, are perfectly valid, and serve to detect metals in biological tissue samples, mainly. However, these methods have their limitations and are not always easily remedied. The method of the present invention has unified the fluorescent techniques and the autometalográficas improving the sensitivity of the second. By increasing the sensitivity levels of a technique, the information collected has a higher quality level.
    [0008] Fluorescence detection is subject to the half-life of the fluorescent complex when it is illuminated to be observed. Autometalogical methods allow the observation of the elements that contain the ion sought during longer periods of time because it is more stable. In any case, there is a problem in the auto-metalogical sensitivity and threshold detection technique. With the method of the present invention using a fluorochrome specific for a metal, an improvement in the sensitivity of existing autoometalography methods is achieved.
    [0009] The use of substances that give color or that emit fluorescence when they come into contact with the divalent metal ions are used to detect them inside the cells; are the so-called "direct methods." The fixation and precipitation of metal ions in the form of precipitates of insoluble salts, their maintenance during the histological process, and their detection after autometalographic development are essential steps for other methods, the so-called "methods". indirect "(such as the methods of Timm and Danscher Neo-Timm).
    [0010] The direct methods can be used in vivo and for quantitative / microanalytical purposes, although they generally do not allow good histological conservation. Indirect methods by contrast, although not very suitable for quantification, are much more sensitive and compatible with optical and electronic microscopy.
    [0011] Indirect methods mainly require that metal ions be precipitated as insoluble nanocrystals.
    [0012] In the state of the art there are known works that disclose detection of various metals by autometalography. For example, the work of Danscher, 1991, refers to the application of autoometalography (AMG) to histological material of humans and animals exposed to gold, silver and mercury, and it has been shown that they can be detected with the optical or electronic microscope. . Danscher, 2002, discloses the detection of gold from implants by autometalography. Danscher, 2008, refers to the detection of mercury present in frogs that capture it from the environment, while Hacker, 1996, refers to autometalography for gold, silver, mercury, bismuth, copper, zinc.
    [0013] Some authors have highlighted the need to detect heavy metals in food (Hosseini, 2015). There are also published works on the detection by autoometalography of metals such as bismuth, which is present in gunpowder (Soltenberg, 2003).
    [0014] Meeusen's work, 2011, also shows an example of the use of TSQ to detect zinc.
    [0015] In the case of zinc, these zinc-specific fluorochromes, and particularly the TSQ, is commonly used for fluorescence, however it has never been used before in zinc autometalography, and its use improves the detection of zinc by autometalography.
    [0016] The zinc that can be detected by histochemical techniques, is the so-called zinc histochemically detectable or labile, and assumes a small fraction of the total amount of zinc in the cell (10-15%). The other forms in which zinc is found, bound to proteins, to enzymes, or to nucleic acids or by means of covalent bonds, are not histochemically detectable.
    [0017] The first reliable method for the histochemical detection of zinc and heavy metals was developed by Timm (1958). A premise of the autometalographic method is that the zinc present in the cells is in ionic form, since most of the detectable zinc accumulates in vesicles, such as synaptic vesicles, secretory vesicles and lysosomes. The two methods widely used to perform this task have been: the addition of sulfur ions in the fixative (Timm and Neo-Timm methods) or the addition of selenium ions (Danscher selenitoj method in vivo immediately before fixation.) It is very important that both precipitates of sulfur or selenium nanocrystals remain insoluble during the histological process. Then, these particles of the metal nanocrystal in sections of tissue are revealed by autometalography. The advantages of this technique are: the high sensitivity, the precise location and the possibility of detecting the metal that is being amplified (Danscher and Stoltenberg, 2006).
    [0018] The autoometalography of silver is based on the deposit of metallic silver on the surface of the metallic nanocrystal and thus be part of it, reducing the silver ions in the solution by capturing the electrons released by the hydroquinone, in this way a metallic crystal is formed. it increases in size progressively over time as long as ionic silver and hydroquinone are available in the solution. This metallic silver envelope around the catalytic center grows until it can be detected by optical or electronic microscopy. It has been calculated that a single gold atom, two silver atoms, and two or three zinc atoms are necessary to act as an autometalographic catalytic center. On the other hand, sulphide deposits (Timm, 1958), selenium complexes (Danscher, 1982), gold, mercury, metallic silver (Danscher et al., 1994), and even SH groups of proteins can act as centers catalytic for autoometalography.
    [0019] Although Timm's method is not specific for a defined metal ion, the use of different treatments of acid samples (dissolving nanocrystals of a particular metal) makes it suitable for microscopic studies of zinc, iron and copper (Kozma et al. , 1981).
    [0020] Modifications of Timm's original technique have allowed the study of zinc in biopsies of human brains by autometalographic detection (Jaarsma and Korf, 1990).
    [0021] The use of different solutions, such as glutaraldehyde saturated with sulfur (Grzybek et al, 1990) or an initial infusion with a solution of sodium sulphide before glutaraldehyde (Ugarte and Osborne, 1998), as well as improvements in the revealing mixture (Danscher and Zimmer, 1978) have allowed Timm's development to be suitable for electron microscopy studies. Thus, electron-dense autogenous silver granules were located within the synaptic vesicles of rat hippocampal neurons (Haug, 1967). And later this finding was confirmed with the sulfhídrico-osmio method (López-García et al., 1984) and the Neo-Timm method (Pérez-Clausell and Danscher, 1985).
    [0022] The classic preparation of samples for zinc autoometalography (Danscher, 1981), includes the precipitation of ionic zinc with sodium sulfide and consists of the following steps:
    [0023] - fixation of the cells with paraformaldehyde (PFA) 4%, glutaraldehyde (GA) 4% or with PFA-GA 1%; 20 minutes at 4 ° C
    [0024] - incubation, for 30 minutes at 4 ° C, with the NTS solution:
    [0025] - 0.1 g of Na 2 S
    [0026] - 6 ml of GA 50%
    [0027] - 50 ml of 0.2 M phosphate buffer pH 7.4
    [0028] -44 ml of distilled water
    [0029] - washing with abundant distilled water, being prepared for further development by autometalography, both with optical microscopy and electron microscopy.
    [0030]
    [0031] In contrast to the above, the preparation of the samples for the autoometalography of the metal, such as zinc, according to the present invention, comprises carrying out at least one incubation of the samples with a fluorochrome specific for said metal, for example for zinc , a zinc-specific fluorochrome.
    [0032] An article that refers to both fluorescence and autoometalography and describes the use of TSQ is Nitzan YB 2004. However these fluorescence and autoometalography techniques to date are used separately and are combined for the first time in the method of the invention, which contributes to an improvement of the Timm method and variants, raising the sensitivity of the limits of detection.
    [0033] Autometalogical methods serve, in theory, for any sample, regardless of origin. With the method of the invention, detection is improved, regardless of whether the sample originates from an experiment with a metal, for example zinc, or otherwise.
    [0034] In the specific case of zinc, its detection in tissues can help to diagnose the state of the cells as a result of diseases, injuries or poisoning.
    [0035] With the present invention has been found an advantageous way to process the samples to stabilize and observe the zincosomes - type of endosomes that are formed in cells such as astrocytes in response to the presence of zinc in the extracellular medium - at the end of the whole process , whose last part is the autoometalography of similar to what is seen with fluorescence, minimizing the loss of labeling.
    [0036] The method of the present invention is useful in various technological areas, such as basic research in science. In the specific case of zinc, it has a specific utility in any biological study that has this metal among its interests. And in general it is useful in medicine, toxicology and food contamination for its application to any metal that can be detected by autometalography, among which is for example, zinc.
    [0037]
    [0038] DESCRIPTION OF THE INVENTION
    [0039] The present invention relates to a method for the detection of a metal by autometalography characterized in that it comprises carrying out before the autoometalographic treatment, at least one incubation of a sample containing the metal, with a fluorochrome specific to the metal.
    [0040] The sample can be of any type, in the case of medicine it can be for example, cells, organs or tissues. In the case of zinc, the sample is, for example, a culture of an animal cell.
    [0041] The metal can be any that can be detected by autometalography and for which specific fluorochromes are known. For example, it can be zinc, copper, mercury. Other metals to which the method of the present invention may be applied will be iron, nickel, chromium, manganese, cobalt, lead, cadmium, molybdenum, tungsten, uranium, arsenic, selenium, bismuth, aluminum and lanthanides.
    [0042] Fluorochromes can be small molecules, genetically encoded sensors, or hybrids. In the case of zinc, a zinc-specific fluorochrome, which may be, for example, a sensor based on quinoline, fluorescein, 4-aminonapthalimide, BODIPY, rhodamine, resorufin or cyanine, will be used. Examples of zinc-specific fluorochromes are New Port green (Newport Green DCF127 and Newport Green PDX123), Fura-2, Fluozin-3, and preferably TSQ.
    [0043]
    [0044]
    [0045] Above are shown in order: Newport Green DCF127 (C 43 H 30 CI 2 N 4 O 8 ), Newport Green PDX (C 31 H 21 F 2 N 3 O 3 ), Fura-2 AM (C 44 H 47 N 3 O 24 ), Fluozin-3 AM (C 46 H 44 F 2 N 2 O 20 ) and TSQ (C 17 H 16 N 2 O 3 S).
    [0046] Examples of fluorochromes that can be used for some metals capable of being detected by the method of the present invention are found in "Fluorescent Sensors for Measuring Metals in Living Systems", (Carter K. P., 2014).
    [0047] Fluorochromes suitable for copper are, for example, a fluorochrome based on phenanthroline, of formula
    [0048]
    [0049]
    [0050] It also serves to detect iron, cadmium, lead, mercury or nickel.
    [0051] Another fluorochrome for copper is one based on formula calcein
    [0052]
    [0053]
    [0054] A known fluorochrome of mercury is represented by the formula C33H15ChK2N3O6 and shown below, commercially called "Phen Green dipotassium salt"
    [0055] Incubation of the sample with a specific fluorochrome of a metal, such as zinc, may have a variable duration, and there is no limitation. In addition, the duration of the incubation depends on the particular metal in question. In the case of zinc and by way of example, the duration of an incubation can be between 0.5 seconds and 30 minutes.
    [0056] The concentrations of fluorochromes can be equally variable, and will depend on the particular metal in question. In the case of zinc and by way of example, fluorochrome concentrations between 1 and 100 ^ g / ml can be used.
    [0057] The number of incubations of the sample with the specific fluorochrome of a metal can be variable, and there is no limitation. In the case of zinc, one or more incubations can be carried out, for example in the case of an animal cell culture sample, two incubations of the sample can be carried out with a zinc-specific fluorochrome.
    [0058] In the case of carrying out more than one incubation, for example two, between the two incubations with a fluorochrome of a metal, the method may comprise a step of fixing the samples. This fixing step can be carried out at variable pH values, depending on the sample, the metal etc.
    [0059] More specific embodiments of the method of the invention relate to the detection of zinc by autometalography, and specifically, in animal cell culture samples. But it should be understood that the method can be applied to another metal and to another type of biological samples. Examples of biological samples are samples of organs, tissues, etc.
    [0060] In specific embodiments, for the case of zinc, the stage of fixing the samples is carried out with PFA at pH between 5.8 and 8.2, preferably between 6 and 8.
    [0061] In specific embodiments for the case of zinc, said fixing step for the case of samples of animal cell cultures, can be performed with PFA at a concentration - expressed as grams per 100 mL of final volume of phosphate buffer - for example between 2% and 6%.
    [0062] Furthermore, said step of fixing the samples can be carried out with PFA at a concentration - expressed as grams per 100 mL of final volume of phosphate buffer and at pH between 5.8 and 8.2, preferably at a pH between 6 and 8.
    [0063] In concrete embodiments for the case of zinc the method includes:
    [0064] - incubation of the sample with TSQ,
    [0065] - fixation with PFA at pH between 5.8 and 8.2, preferably at pH between 6 and 8,
    [0066] - incubation of the sample with TSQ.
    [0067] According to additional particular embodiments for the case of zinc autoometalography in animal cell culture, the stage of fixation of the samples is carried out with 4% PFA and pH 7.4.
    [0068] According to further particular embodiments for zinc autoometalography, the sample is cultured astrocytes, and the method comprises:
    [0069] - incubation of the sample with TSQ at 50 ^ g / ml for 10 minutes,
    [0070] - fixation with PFA 4% at pH 7.4,
    [0071] - second incubation of the sample with TSQ at 50 ^ g / ml for 10 minutes.
    [0072]
    [0073] According to additional particular embodiments for zinc autoometalography the method comprises after the second incubation with TSQ:
    [0074] - incubation, for 30 minutes at 4 ° C, with an NTS solution,
    [0075] - washing with abundant distilled water, the samples being prepared for further development by autometalography.
    [0076] According to further particular embodiments for zinc autoometalography, the method comprises:
    [0077] - incubation of the sample with TSQ at 50 ^ g / ml for 10 minutes,
    [0078] - fixation with PFA 4% at pH 7.4,
    [0079] - incubation of the sample with TSQ at 50 ^ g / ml for 10 minutes,
    [0080] - incubation, for 30 minutes at 4 ° C, with an NTS solution,
    [0081] - washing with abundant distilled water, the samples being prepared for further development by autometalography,
    [0082] which represent the optimal conditions for the correct stabilization and detection of zinc by means of autometalogistic methods in tissues and, in general, in any sample.
    [0083] The present invention also relates to the use of the method described for zinc detection in zincosomes.
    [0084]
    [0085] BRIEF DESCRIPTION OF THE FIGURES
    [0086] Figure 1 shows the (known) formation of zincosomes (endosomes containing zinc) in astrocytes with, and without, exogenous zinc pulse.
    [0087] Figure 2 shows the effect of TSQ after fixation on the labeling of zincosomes with Neo-Timm. Specifically, Figures 2 (AE) show astrocytes incubated with different protocols: with and without exogenous zinc, and with, and without, the TSQ marker. In the series of photos only positive marks are seen in the samples that have had some incubation with TSQ: photo C an incubation and E two incubations.
    [0088] Figure 3 shows the effect of the use of TSQ to increase the detection capacity of zinc by autometalography in cultured cells.
    [0089] Figure 4: shows on the one hand the effect of the pH of the fixative on the final result. With a TSQ incubation, only a pH of 7.4 is seen with Neo-Timm. With two incubations, we found brand with any pH.
    [0090] Figure 5: shows the quantification of marked zincosomes after development with Neo-Timm using fixing solutions with different pH values and with one or two incubations with TSQ.
    [0091] The examples shown below refer to zinc autoometalography in biological samples, but as indicated, the method can be applied to other metals and to other types of samples.
    [0092]
    [0093] EXAMPLES
    [0094] Example 1
    [0095] Reagents used in detail with their concentrations and methods of preparation:
    [0096] Fixing solutions (100 ml):
    [0097] • Paraformaldehyde (PFA), stock solution (8%):
    [0098] - 8 g of PFA (818715, Merck).
    [0099] -100 ml distilled water.
    [0100] - Shake on a hot plate until it dissolves.
    [0101] - Immerse a NaOH lentil (141687, Panreac) for a few seconds until the solution becomes clear.
    [0102] - Allow to cool, aliquot and freeze until use.
    [0103] • Paraformaldehyde 4% (PFA 4%):
    [0104] - 50 ml of PFA 8% stock solution.
    [0105] - 50 ml of 0.2 M phosphate buffer pH 7.4.
    [0106] • 4% Glutaraldehyde (GA 4%):
    [0107] - 8 ml of GA 50% (15A807, Panreac).
    [0108] - 50 ml of 0.2 M phosphate buffer pH 7.4.
    [0109] - 42 ml of distilled water.
    [0110] • Paraformaldehyde 1% and glutaraldehyde 1% (PFA 1% -GA 1%):
    [0111] -2 ml of GA 50%.
    [0112] - 12.5 ml of PFA 8% stock solution.
    [0113] - 50 ml of 0.2 M phosphate buffer pH 7.4.
    [0114] - 35.5 ml of distilled water.
    [0115] Postfixing solutions:
    [0116] • NTS (Neo-Timm solution, Danscher, 1981)
    [0117] - 0.1 g of Na 2 S (208043, Sigma-Aldrich).
    [0118] - 6 ml of GA 50%.
    [0119] - 50 ml of 0.2 M phosphate buffer pH 7.4.
    [0120] - 44 ml of distilled water.
    [0121] Tampons used:
    [0122] • Phosphate buffer (PB), stock solution (0.2 M) pH 7.4:
    [0123] 22.72 g of Na 2 HPO 4 (MW 141.96 g / mol) (106586, Merck).
    [0124] - 4.8 g of NaH 2 PO 4 (MW 120 g / mol) (106370, Merck).
    [0125] - Dissolve in distilled water and make up to 1 liter.
    [0126] • Phosphate buffer (PB), working solution (0.1 M) pH 7.4:
    [0127] - Mix in a 1: 1 ratio, 0.2 M PB buffer and distilled water.
    [0128] • Saline phosphate buffer (PBS), working solution (0.01 M) pH 7.4:
    [0129] - 50 ml of 0.2 M phosphate buffer pH 7.4.
    [0130] - 9 g NaCl (27800, Rectapur).
    [0131] - Add distilled water and make up to 1 liter.
    [0132] Revealing solution of Danscher Neo-Timm (100 ml):
    [0133] - 60 ml of 50% gum arabic in distilled water (142061, Panreac).
    [0134] - 10 ml of 2 M citrate buffer pH 3.7, prepared with 25.5 g of citric acid 1-hydrate (141018, Panreac) and 23.5 g of tri-sodium citrate 2-hydrate (131655, Panreac) and make up with distilled water up to 100 ml.
    [0135] -15 ml of Hydroquinone 5.7% in distilled water (822333, Merck).
    [0136] -15 ml of Silver Lactate 0.73% pH 3.8 in distilled water (L7771, Sigma-Aldrich).
    [0137] Example 2
    [0138] Tests with different fixing solutions with different pH value.
    [0139] Different buffer solutions were prepared with the different pH values tested (6.0, 7.4 and 8.0), so that when adding the fixative PFA 4% we had fixing solutions with different pHs.
    [0140] It was observed - as can be seen in figure 4 and in figure 5 - as the fixing solution with pH 7.4 was the only one showing zincosomes marked with a TSQ incubation and the one with the most zincosomes marked with two TSQ incubations with respect to the other pH values (6.0 and 8.0) after the autometalographic development.
    [0141]
    [0142] Tests with different fixing solutions.
    [0143] For this, different fixative solutions were prepared, PFA 4%, GA 4% and PFA1% -GA1%.
    [0144] It was observed how the fixative solution PFA 4% was the one that more zincosomas marked after the autometalogical development.
    [0145]
    [0146] Example 3
    [0147] Tests with different incubation times with NTS.
    [0148] To check the incubation time with NTS that gave better results, cells were incubated with NTS for 3 times: 30 minutes, 3 hours and 24 hours. It was observed how the NTS solution applied for 30 minutes was the one that showed the most zincosomes after the autometalographic development.
    [0149]
    [0150] Example 4
    [0151] Self-filtering or Neo-Timm development
    [0152] Autometalogical development or Neo-Timm was performed in all cases after incubation procedures with TSQ, fixation and incubation with NTS, is the last step of the method. And it was carried out in the following way in which the numerical sequence follows the chronological order:
    [0153] Pre-treatment to detect captured zinc. In this particular example, zinc is added to evaluate its uptake by astrocytes. Therefore, for this specific case these are previous steps to study the uptake of zinc in astrocytes. Put TPEN to remove the pre-existing zinc and then add zinc. In other types of samples, these steps would not be necessary:
    [0154] 1- Incubation with 25 ^ M TPEN (N, N, N ', N'-tetrakis (2-pyridylmethyl) ethylenediamine) (T1210, Life Technologies) in DMEM base (DMEM: Dubelcco's Modified Eagle Medium) for 20 minutes at 37 ° C.
    [0155] 2- Wash the cells with DMEM base.
    [0156] 3- Incubation with 50 ^ M ZnSO 4 in DMEM base for 10 minutes at 37 ° C.
    [0157] 4- Wash the cells with DMEM base.
    [0158] Steps for improving the sensitivity of autoometalography according to the invention: 5- Incubation with 152 ^ M (50 ^ g / ml) TSQ in DMEM base for 10 minutes at 37 ° C.
    [0159] 6- Fixation with PFA 4% at pH 7.4 for 30 minutes at 4 ° C.
    [0160] 7- Incubation with TSQ (50 ^ g / ml) 10 minutes (convenient and necessary in samples with pH other than 7.4).
    [0161] 8- Incubation with NTS 0.1% and GA 3% in PB buffer 0.1 M pH 7.4 for 30 minutes at 4 ° C.
    [0162] 9- Wash with distilled water.
    [0163] Development steps, or autoometalography itself:
    [0164] 10- Immerse the cells in the autometalographic solution at 37 ° C in the dark for approximately 30 minutes.
    [0165] 11- Wash with abundant distilled water to stop the development reaction and completely eliminate the development solution.
    [0166] 12- Stabilize the metallic silver by immersing the cells in a 10% (w / v) sodium thiosulfate solution for 10 minutes at room temperature.
    [0167] 13- Wash the cells with plenty of distilled water.
    [0168] 14- Mount the preparations with some medium that does not dissolve the formed precipitate (for example, Mowiol 4-88 (17951, Polysciences)).
    [0169]
    [0170] Example 5
    [0171] Test for detection of zincosomes
    [0172] Figure 1 shows the formation of zincosomes (endosomes containing zinc) with exogenous zinc pulse. The differences between cultures of astrocytes incubated with, or without, an exogenous zinc pulse, which have been previously incubated with TPEN (N, N, N ', N'-tetrakis (2-pyridylmethyl) ethylenediamine) to eliminate the free zinc, and this exogenous zinc having been subsequently labeled with TSQ.
    [0173] In image (A) the astrocytes were incubated with 50 ^ M ZnSO 4 for 10 minutes and in (B) only with fresh medium. The term "new medium" refers to a nutrient medium commercial. The cells in culture are always kept submerged in a culture medium containing water, mineral salts, glucose etc; necessary for its maintenance. If we add zinc to the cells with the culture medium, later we can see fluorescent spots; if we add culture medium without zinc (= new medium), we will not see fluorescence. Therefore, it can be deduced that the observed fluorescence is due to the zinc added, not to the nature of the cells or the culture conditions.
    [0174] When the cells were incubated with the TSQ marker, we observed that only the cells that had received the exogenous zinc pulse showed bright zincosomes (A), whereas the cells without the zinc pulse showed no type of mark (B). Scale: 20 ^ m.
    [0175]
    [0176] Essay to show the importance of the TSQ fluorochrome
    [0177] The importance of the use of TSQ to increase the capacity of zinc detection by autometalography in cultured cells was also analyzed, which is shown in figure 2. The effect of TSQ after fixation on the labeling of zincosomes with Neo-Timm was analyzed. . Thus, Figure 2 (AE) shows a representative image of the differences showing astrocytes incubated with different protocols: with (concentration in the incubation medium ZnSO 4 : 50 ^ M) and without exogenous zinc, and with (concentration in the TSQ incubation medium: 50 ^ g / ml), and without, the TSQ marker. In the example, exogenous zinc is added to ensure that zinc exists in the test cells and to demonstrate what is intended to be achieved with the present invention, that is, the technical effect thereof, which is the improvement of the method with the (s) incubation (s) with the zinc-specific fluorochrome, and especially TSQ.
    [0178] In Figures 2A-2E, the symbol of the empty set means that no substance is added during the treatment.
    [0179] That is, the samples receive 4 treatments. Some samples are zinc, TSQ, while others, at a specific time, do not put that substance. This has compared the effect of various additions with respect to not making those additions.
    [0180] Subsequently, the zinc was fixed and precipitated with NTS before revealing the monolayers with Neo-Timm. We can see how without zinc pulse, zincosomes are not formed with TSQ (A), nor without TSQ (B). The cells that were incubated with zinc, showed zincosomes marked by autometalography after incubating them with TSQ (C), but without the incubation with the fluorochrome, no mark (D) was obtained. The monolayers that were incubated with zinc, TSQ and fixed, were incubated again with TSQ before the development of Neo-Timm, these showed a density of marked zincosomes greater (E) than the monolayers without the second incubation of TSQ after fixation (C ). Scale: 20 ^ m.
    [0181]
    [0182] Effect of the use of TSQ to increase the detection capacity of zinc by autometalography in cultured cells
    [0183] The effect of the use of TSQ to increase the capacity of zinc detection by autometalography in cultured cells was also studied (analysis and quantification of images shown in the same conditions as in Figures 2A to 2E). Thus, it is observed in figure 3 how, the cells without the incubation with the TSQ marker, after the autometalographic development did not show any type of mark. When the live astrocytes were incubated with TSQ, after the development we marked the zincosomes with metallic silver, thus being quantifiable. Finally, the monolayers that were incubated a second time with the TSQ fluorochrome after the fixation, marked the double zincosomes after the autometalographic development that with a single incubation with TSQ. The asterisks indicate significant differences (Student's t, *** p <0.001, n = 174 cells).
    [0184]
    [0185] Effect of the use of TSQ to increase the detection capacity of zinc by autometalography even in non-physiological conditions
    [0186] The effect of using TSQ to increase zinc detection capacity by autoometalography in non-physiological conditions was also studied. Figure 4 shows the effect of TSQ after fixation on the labeling of zincosomes with Neo-Timm and also shows the effect of pH on the labeling of zincosomes with Neo-Timm.
    [0187] Astrocytes were incubated with TPEN, zinc and TSQ. Next, the monolayers were fixed with 4% PFA with different pH values to see if this affected the conservation and detection of the ionic zinc. Some cells were incubated again with TSQ. Finally, the monolayers were fixed with NTS and revealed with the Neo-Timm method. We observed that with a single incubation with TSQ (A, C and E), only monolayers fixed with 4% PFA at pH 7.4 show marked zincosomes. However, all monolayers with two incubations of TSQ (B, D and F), show zincosomes marked by the cytoplasm, with PFA at pH 7.4 being the most marked zincosomes. Scale: 20 ^ m.
    [0188] Furthermore, in Figure 5, which represents the analysis and quantification of the experiments corresponding to a set of images as shown in Figure 4, the importance of the use of TSQ to increase the detection capacity of zinc by autometalography is illustrated, even in non-physiological conditions. Thus, the effect of pH on the labeling of zincosomes with Neo-Timm can be observed.
    [0189] The graph of figure 5 shows the density of marked zincosomes after development with Neo-Timm using fixing solutions with different pH values and with one or two incubations with TSQ. We see how the PFA fixative at pH 7.4 presents the highest density of zincosomes, both with an incubation of TSQ and with two, in comparison with the other two fixing solutions pH 6.0 and pH 8.0. Monolayers with a single incubation with TSQ only show zincosomes labeled with pH 7.4 and have a significantly lower zincosome density with respect to monolayers with double incubation with TSQ with all pH values of the fixative. The asterisks indicate significant differences (Student's t test, *** p <0.001, * p <0.05, n = 120 cells).
    [0190] In order to study the zincosomes with indirect methods and thus avoid the drawbacks of fluorescence, such as the extinction of the mark with the illumination of living cells, the best conditions for the fixation of monolayers of astrocytes were determined. The objective was to preserve the zinc as best as possible for its subsequent detection with autoometalography and even electron microscopy.
    [0191] In the fluorescence analysis, we saw how all the fixing solutions (process necessary for the autoometalography process) reduced the fluorescence levels. They also reduced the density of endosomes that form in astrocytes by incubating them in the presence of zinc, called zincosomes. But it was the PFA 4% that best preserved the fluorescence of the TSQ with respect to the fluorescence levels observed in living cells.
    [0192] The first results (corresponding to the experiments in figure 1 and 2) show us that incubation with TSQ prior to fixation is a determining factor to obtain results similar to what is observed with living cells.
    [0193] We also check that the pH of the fixative is a determining factor to be taken into account when fixing the cells, since zinc is a very labile ion and it can be lose samples easily with both fixation and washing. Previously we saw that the fixative prepared with buffer at pH 7.4 (physiological conditions) is the one that best preserves the zinc with the fluorescence of TSQ, with a smaller reduction in the density, brightness and size of the zincosomes that the fixers prepared with buffer at pH 6 and 8 (data not shown because they do not affect the purpose of the patent).
    [0194] The second fixation with TSQ results in a greater autometalographic marking in all cases. This may be due to the fact that the zincosomes have lost part of their zinc content with the fixation and the washes, and it is in the smaller zinc accumulations, where this loss is more noticeable, up to levels below the limit of detection. Therefore, this second incubation with TSQ serves to verify that the zincosomes of the cells maintain zinc in their interior, and at the same time to be able to determine the presence of zinc in a more appropriate way, with greater sensitivity. It is understood that the TSQ of the second incubation after fixation, binds to zinc that has endured the process of fixation and post-fixation, and acts as a mordant in the development process of Neo-Timm, thus improving the results of autoometalography , regarding the state of the art.
    [0195]
    [0196] Bibliography
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    权利要求:
    Claims (13)
    [1]
    A method for the detection of a metal by autometalography in a sample of biological tissue, characterized in that it comprises carrying out at least one incubation of the sample with a specific fluorochrome of said metal.
    [2]
    2. The method according to claim 2, characterized in that the sample is a cell culture
    [3]
    3. The method according to claim 2, characterized in that the sample is an animal cell culture.
    [4]
    The method according to one of the preceding claims, characterized in that it comprises carrying out two incubations with the fluorochrome, and between the two incubations it comprises a step of fixing the samples.
    [5]
    The method according to one of the preceding claims, characterized in that the metal is zinc, copper or mercury.
    [6]
    6 The method according to claim 5, characterized in that the metal is zinc and comprises carrying out at least one incubation of a sample with a zinc-specific fluorochrome.
    [7]
    The method according to claim 6, characterized in that the metal is zinc and that the stage of fixing the samples is carried out with PFA.
    [8]
    8 The method according to claim 7, characterized in that the PFA is in a concentration between 2% and 6%.
    [9]
    The method according to one of claims 7 or 8, characterized in that the step of fixing the samples is carried out with PFA at pH between 5.8 and 8.2.
    [10]
    The method according to claim 6, characterized in that the zinc-specific fluorochrome is the TSQ.
    [11]
    The method according to one of claims 6 to 10, characterized in that it comprises:
    - incubation of the sample with TSQ,
    - fixation with PFA between 5.8 and 8.2.
    - incubation of the sample with TSQ.
    [12]
    12. Use of the method defined in any one of the preceding claims in the autometalographic technique.
    [13]
    13. Use of the method according to claim 12 in biological studies for medical purposes, for toxicology or related to food contamination.
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