LABELING OF ALUMINOSILICATES

Abstract

Labeling and detection of clinoptilolite and other zeolites and aluminosilicates by means of lumogallion fluorescence reaction in paraformaldehyde-fixed animal and human cell cultures and tissue samples after administration of the mineral or in mineralogical-geological samples themselves.

Claims

1-7. (canceled)

8. A method for the specific fluorescence labeling of aluminosilicates in a sample, comprising: incubating the sample with a lumogallion (5-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzenesulfonic acid) solution in a buffer having a pH value ranging from pH 7.2 to pH 4.0; wherein incubating the sample includes either incubating the sample for a period of from 2 to 4 hours at a temperature of 60 to 80 C., or incubated the sample for a period of time from 12 to 24 hours at a temperature of 20 to 30 C.; and stabilizing the sample using a mounting medium.

9. The method of claim 8, wherein the specific fluorescence labeling of aluminosilicates in the sample includes the specific fluorescence labeling of zeolites in the sample.

10. The method of claim 8, wherein the specific fluorescence labeling of aluminosilicates in the sample includes the specific fluorescence labeling of clinoptilolites purified from heavy metals in the sample.

11. The method of claim 8, wherein the specific fluorescence labeling of aluminosilicates in the sample includes specific fluorescence labeling of aluminosilicates in a biological sample.

12. The method of claim 8, wherein the specific fluorescence labeling of aluminosilicates in the sample includes specific fluorescence labeling of aluminosilicates in a cell sample.

13. The method of claim 8, wherein the specific fluorescence labeling of aluminosilicates in the sample includes specific fluorescence labeling of aluminosilicates in a histological sample.

14. The method of claim 8, wherein the specific fluorescence labeling of aluminosilicates in the sample includes specific fluorescence labeling of aluminosilicates in a sample embedded in either a paraffin or a paraffin substitute.

15. The method of claim 14, wherein labeling the sample includes labeling the sample without dewaxing the sample.

16. The method of claim 8, wherein the specific fluorescence labeling of aluminosilicates in the sample includes specific fluorescence labeling of aluminosilicates in a mineralogical sample or a geological sample, and where the sample was embedded in either a paraffin or a paraffin substitute.

17. The method of claim 8, wherein the specific fluorescence labeling of aluminosilicates in the sample includes specific fluorescence labeling of aluminosilicates in an in vitro-cell culture sample; and further comprises incubating the in vitro-cell culture sample with aluminosilicates or with mixtures of aluminosilicates and non-aluminosilicates; and fixing the sample; before treating the fixed sample with the lumogallion solution.

18. The method of claim 8, wherein the specific fluorescence labeling of aluminosilicates in the sample further comprises an analytical-diagnostic rapid detection of the labeled sample, or an imaging of the sample.

19. The method of claim 8, wherein stabilizing the sample using a mounting medium includes stabilizing the sample using a mounting medium that includes para-phenylenediamine (1,4-diaminobenzene) in glycerol.

Description

[0048] The attached drawing represents in:

[0049] FIGS. 1, 2, 4, 5, and 6 examples for the staining and in FIG. 3 an example without evaluable staining, and are explained in more detail below.

[0050] More specifically, it is advantageous for the application of the labeling of the alumosilicates alone or in biological specimens by means of lumogallion, both for the duration and the incubation temperature, as well as the concentrations, to keep pH values of the buffers and solutions used as well as the particle number in accordance with the following schedule: [0051] a. Zeolites or other aluminosilicates: 10 g/cm.sup.2 [0052] b. Diameter of particle size: approx. 1-60 m [0053] c. Lumogallion solution: 10-200 M in 50 mM HEPES buffer pH 7.2 or 100-200 M in 20-50 mM acetate buffer, pH 4.0. [0054] d. Incubation period: 2 h-24 h [0055] e. Incubation temperature: 20 C.-37 C./50 C.-80 C.

[0056] On an experimental scale, the fluorescence reaction may be carried out at (room) temperature of up to 37 C. for 12 to 24 hours (overnight) as well as at 50 C. to 80 C. within 2 to 4 hours.

[0057] Since labeling using lumogallion is known to be non-toxic, it is also possible for the experimenter to handle the solutions and the process in a comfortable and unproblematic manner while adhering to all protective measures required for working in a laboratory and with the required substances.

DETAILED DESCRIPTION

Cell Culture Monolayers:

[0058] In paraformaldehyde-fixed cell cultures, which were previously treated with aluminosilicates or aluminosilicates in mixtures with non-aluminosilicates, aluminosilicates can be definitely detected. [0059] Any autofluorescence of the cells is surprisingly reduced by treatment with lumogallion and the labeled particles are unmistakably recognizable. [0060] A particular advantage of this specific labeling of aluminosilicate particles using lumogallion is the surprisingly high stability of fluorescence, which can be further extended using an antioxidative mounting medium, so that even after several days to months of storage at 4 C. the samples remain analyzable under a microscope without significant loss of quality. FIGS. 1 and 5, as well as 6b depict several examples of this application.

Tissue Samples:

[0061] Histological sections of paraffin-embedded, zeolite-containing samples (with a thickness of 5 m-25 m) may surprisingly also be stained with a buffered lumogallion solution without prior dewaxing. [0062] As with cell culture monolayers, autofluorescence of the tissue layer is also suppressed (quenched) and the zeolites become specifically visible. [0063] A peculiarity of this method lies in the surprisingly high stability of the labeling and physicochemical integrity of the fluorophore (lumogallion) after fixation and embedding, which can additionally be carried out using an antioxidant mounting medium. This counteracts any possible bleaching of the fluorophore by the excitation light during analysis using fluorescence microscopy (photobleaching). It was discovered that the samples are observable for a longer period of time without significant loss of fluorescence under the microscope and that they can be stored refrigerated at 4 C. (for several days to months). [0064] FIG. 5 shows an analysis of samples which had been stored for 8 weeks in a refrigerator at 4 C. after lumogallion staining and whose fluorescence label is still clearly delineated and intensively bright after this period without any significant loss of quality.

Mineralogical Geological Samples:

[0065] The labeling of aluminum in aluminosilicates not only allows the detection of zeolites in biological samples, but also to differentiate them in rock samples from other minerals using lumogallion stainingalbeit surprisingly only after embedding them in paraffin/paraffin substitute. This embedding turns out to be a decisive and unavoidable step, as well as a step never described so far in the detection process (see FIG. 2), without which there is no evaluable labeling with lumogallion (see FIG. 3). A summary of examples of various rocks showing staining or no staining with lumogallion can be found in FIG. 6a. [0066] The staining of aluminum with lumogallion offers the advantage of being feasible under physiological pH (7.0-7.2) and using conventional (cell culture) buffers (such as HEPES or PIPES) and media. Alternatively, the specimens may also be incubated in the acidic range and with other conventional buffers such as 20 mM acetate buffer, pH 4.0. The wide range of buffers and pH values is extremely useful in practical applications. [0067] To detect fluorescence resulting from the reaction of lumogallion with aluminum from the sample, no special equipment dedicated to this purpose is necessary, but commercially available epifluorescence and confocal microscopes and spectrophotometers with the appropriate filters can be used. [0068] Due to the relatively high stability of the labeling after fixation and embedding using a mounting medium, the samples can be examined or stored under a microscope for longer periods of time (several days to months) without significant loss of quality, which is particularly characteristic of the invention (see e.g. FIG. 5).

[0069] The invention will be described in more detail below, wherein the individual Figures show the following:

[0070] FIG. 1Lumogallion staining of zeolite in fixed cell cultures

[0071] Evaluation of the samples was carried out on an epifluorescence microscope using special filters (see Table 2).

[0072] The excitation took place at about 500 nm, the emission was detected at around 570 nm.

[0073] FIG. 2Joint embedding of different particles (zeolite and activated carbon or zeolite and silicon) followed by lumogallion staining

[0074] Joint embedding of different rock samples showing either fluorescence staining or no fluorescence staining with lumogallion.

[0075] Both the specificity of lumogallion labeling for aluminum-containing compounds as well as the specificity of the individual filters for different rocks become apparent.

[0076] FIG. 3Particle staining with lumogallion without prior embedding

[0077] Staining of zeolite particles was carried out without prior embedding in paraffin/paraffin substitute.

[0078] No labeling could be detected in the sample.

[0079] FIG. 4Lumogallion-stained histological samples

[0080] Paraffin-embedded, non-dewaxed intestinal tissue samples from mice fed with and without zeolite admixture in the diet after staining with lumogallion.

[0081] FIG. 5Zeolite-treated, fixed and lumogallion-labeled cells which had been stored for 8 weeks at 4 C.

[0082] After the first analysis, the samples were kept unchanged in the refrigerator at 4 C. for 56 days before being re-evaluated.

[0083] FIG. 6Fluorescence images of the test substances summarized in Table 1

[0084] Overview of various rock specimens embedded in paraffin substitute (Paraplast) or used for cell culture treatments, which stained positively with Lumogallion or could not be labeled with Lumogallion.

[0085] In FIGS. 1-5, clinoptilolite (purified according to U.S. Pat. No. 8,173,101 B2) was used; this is an aluminosilicate of volcanic origin and belongs to the group of zeolites within the tectosilicates.

[0086] FIG. 6 allows a comparison of clinoptilolite with the artificially produced HY zeolite both when used in cell cultures and after embedding in paraffin substitute (Paraplast).

[0087] In detail, the figures represent:

[0088] FIG. 1 shows a typical analysis with an epifluorescence microscope with 100 magnification (Axiovert 200M, ZEISS company) of an application of the lumogallion staining of paraformaldehyde-fixed human cell cultures (MCF-10A, ATCC CRL-10317) with (FIG. 1a) and without (FIG. 1b) clinoptilolite treatment. Clinoptilolite is an aluminosilicate of volcanic origin and belongs to the group of zeolites within the tectosilicates. Prior to this, the samples were incubated overnight at 37 C. with 100 M lumogallion in 50 mM HEPES buffer, pH 7.2. In order to minimize fading of fluorescence staining, an antioxidant mounting medium (VECTASHIELD Antifade Mounting Medium, Vector Laboratories, USA) was used. The filters described in Table 2 were used for the analysis. Clinoptilolite particles fluoresce clearly andusing the FITC filter (FIG. 1a)can even be recognized individually. The untreated clinoptilolite particles of the control show no autofluorescence (FIG. 1b). The scale bar corresponds to 20 m.

[0089] FIG. 2 depicts joint embedding of clinoptilolite with particles which are not stainable with lumogallion. For this purpose, the particles (clinoptilolite and activated carbon, FIGS. 2a and 2b, or clinoptilolite and silicon, FIGS. 2c and 2d) were embedded in paraffin substitute (Paraplast X-tra Tissue Infiltration/Embedding Medium, McCormick Scientific, PA) and subsequently Mictrotome-sectioned at room temperature with a specimen thickness of 16 m. For staining with 100 M lumogallion in 50 mM HEPES buffer, pH 7.2, the section was incubated overnight at 37 C. (FIGS. 2a and 2c). FIGS. 2b and 2d show the associated controls which were treated the same as the samplesexcept that they were incubated overnight in 50 mM HEPES buffer without lumogallion. Both controls and samples were protected from bleaching with mounting medium (VECTASHIELD Antifade Mounting Medium, Vector Laboratories, USA). The samples used in FIGS. 2a and 2b as well as 2c and 2d are each immediately successive serial sections with different areas shown in the figures. As can be clearly seen in FIGS. 2a and 2c, an evaluation using an epifluorescence microscope with 100 magnification (Axiovert 200M, ZEISS), and in the case of the activated carbon-clinoptilolite mixture an evaluation with all the filters described in Table 2, is possible without difficultythe staining of clinoptilolite is vibrant while the activated carbon does not fluoresce. The situation was similar with a barium sulfate-clinoptilolite mixture (not shown). However, the silicon-clinoptilolite mixture could only be analyzed with the FITC and TRITC filters, since the excitation in the UV light range required for evaluation using morin filters heated the silicon particles, causing the Paraplast to melt and the particles to begin to diffuse, which made detection impossible. This impressively illustrates the high specificity of the labeling of aluminum using lumogallion, as well as the need to use suitable filters which are to be chosen specifically for the individual samples. The scale bar corresponds to 20 m.

[0090] FIG. 3 represents particle staining of clinoptilolite without prior embedding in paraffin or Paraplast in brightfield and using different filters (FITC, TRITC, Morin). The samples were incubated with a particle concentration of 133 g/ml with 100 M lumogallion in 50 mM HEPES buffer, pH 7.2 at 37 C. overnight, then centrifuged, and the pellet was fixed in 15 l mounting medium (VECTASHIELD Antifade Mounting Medium, Vector Laboratories, USA) on a microscope slide. Labeling of the clinoptilolite particles with lumogallion is hardly detectable with an epifluorescence microscope with 100 magnification (Axiovert 200M, ZEISS) and therefore can not be evaluated. The scale bar corresponds to 20 m.

[0091] FIG. 4 illustrates murine, non-dewaxed, histological intestinal samples of a clinoptilolite-fed mouse (FIG. 4a) and a control mouse without clinoptilolite admixture in the feed (FIG. 4b). Tissue sections were imaged with a 32 magnification epifluorescence microscope (Axiovert 200M, ZEISS), first untreated (FIGS. 4a and 4b BEFORE) and subsequently stained with lumogallion (FIGS. 4a and 4b AFTER). The scale bar corresponds to 50 m. After sampling, fixation, and subsequent paraffin embedding, the samples were stored at room temperature for about 2.5 years before being sectioned (to approximately 15 m) and used for lumogallion staining. The sections were incubated overnight at 37 C. with 100 M lumogallion in 50 mM HEPES buffer, pH 7.2, and then fixed on a microscope slide using mounting medium (VECTASHIELD Antifade Mounting Medium, Vector Laboratories, USA). The staining of the particles is clearly visible (FIG. 4a AFTER). The long storage period of histological sections did not minimize the quality of lumogallion labeling of clinoptilolite particles. FIG. 4b AFTER illustrates the quenching of autofluorescence by incubation with lumogallion.

[0092] FIG. 5 shows two examples of fluorescence images of an evaluation of samples labeled with lumogallion after a longer storage period (8 weeks at 4 C.). Human cell cultures (MCF-10A, ATCC CRL-10317) were fixed with paraformaldehyde following clinoptilolite treatment and subsequently incubated with lumogallion. After initial analysis, the clear-coated samples remained unopened for 2 months under continuous cooling without further manipulation, before additional analysis (FIG. 5) was performed. Evaluation was carried out at 100 magnification (Axiovert 200M, ZEISS) using the FITC filter. Clinoptilolite particles continue to fluoresce intenselyfluorescence does not appear to be noticeably reduced due to the long storage period. 100 magnification. The scale bar corresponds to 20 m.

[0093] FIG. 6 shows a summary of the test substances used and their reaction to lumogallion incubation. While FIG. 6a shows the particles embedded in Paraplast (paraffin substitute), FIG. 6b summarizes cell cultures incubated with aluminosilicate or non-aluminosilicate particles, then fixed and finally treated with lumogallion. The samples were always evaluated under the same conditions (FITC/TRITC/morin filter, Axiovert 200M, ZEISS) and using the same magnification (100). The intensity of the staining is indicated as follows: ++ means strong, + means weak and means no labeling of the particles with lumogallion. The symbol / was used to indicate experimental problems, which are further explained in the Notes column; in the examples given, it specifically was a strong autofluorescence of the test substance.

[0094] Specific detection of aluminosilicate compounds in cells (FIGS. 1, 5, and 6b) and tissues (FIG. 4) enables the identification and distribution of the same, and thus for the first time, detection and tracking of these particles after their application. Of great advantage is the possibility of subsequent labeling of aluminosilicates in biological material, since the lumogallion reaction is also feasible in paraformaldehyde-fixed preparations without loss of quality of the label. Dewaxing of samples embedded in paraffin/paraffin substitute is surprisingly unnecessary for successful staining.

[0095] Furthermore, staining with lumogallion surprisingly quenches any autofluorescence of the cells or the cell structure (FIG. 4b AFTER), which further contributes to a distinct identification of the zeolite particles.

[0096] Labeling of the particles surprisingly proves to be very stable, so that these are clearly visible even days or months later (FIG. 5).

[0097] The ability to label zeolite particles over a broad temperature range and at variable pH values significantly expands the application spectrum and contributes to a diverse application.

[0098] Staining of aluminosilicate particles with only lumogallion is also possible (FIG. 6a)but surprisingly only if these were previously embedded in paraffin or paraffin substitute (see FIG. 3, which shows no staining of particles with lumogallion without paraffin or Paraplast). As a result, zeolites can also be distinguished from other substances or mineralsexamples being joint embedding of clinoptilolite and activated carbon (FIGS. 2a and 2b), or clinoptilolite and silicon (FIGS. 2c and 2d), or also clinoptilolite and barium sulfate (not shown), wherein in each case only clinoptilolite was stained, while activated carbon (FIG. 2a), silicon (FIG. 2c), and barium sulfate respectively remained non-fluorescent.

[0099] If necessary, re-staining of bleached particles is possible without restriction and useful, if previously labeled samples need to be analyzed again. To this end, the samples are briefly rinsed three times in the buffer in which the subsequently used lumogallion solution was prepared (e.g., HEPES or acetate buffer) and then incubated in the lumogallion labeling solution. Incubation time and temperature, as well as the further procedure up to and including embedding may correspond to those of the primary incubation (see embodiments).

[0100] In addition, labeling with lumogallion has the advantage of being detectable over a broad spectrum so that the most common filters (see Table 2) may be used. In this case, it is recommended to test the filter most suitable for each respective test seriesminerals or rocks have different fluorescence maxima/fluorescence spectra with lumogallion staining and the choice of filters should be taken into consideration in this regard (for example FIG. 2cin this case, silicon particles cannot be detected with a morin filter).

[0101] The user can work with the usual equipment of a cell biological laboratory; the acquisition of specialized equipment produced solely for lumogallion staining is unnecessary.

[0102] In summary, the invention is therefore characterized by its rapid and uncomplicated practicability, its specificity for aluminosilicates, its high reproducibility and stability over several days or months, as well as by the elimination of dewaxing of histological specimens and the possibility of renewed/repeated staining with lumogallion.

[0103] This characteristic may prove particularly useful in forensic analysis of soil samples (Tibet and Carter, 2008). It is also possible to identify aluminosilicates in human and animal histospecimens which were created in the distant past and may thus yield new insightswithout additional larger investments of money and time.

[0104] Furthermore, it is possible to identify aluminosilicates in mineral mixtures/rock mixtures in an equally specific manner and also, as a consequence, to define the amount of aluminosilicates in the substance to be tested.

[0105] Within a short period of time and with little technical resources and required staff, this method achieves a highly reproducible detection of aluminosilicates in the context of analytical-diagnostic quick detection or for visualization using fluorescence microscopy and the appropriate filter.

Examples of Embodiments

Example of Labeling of Aluminum in Cell Culture Samples:

Sample Preparation:

[0106] Various rock samples (see Table 1) were diluted in growth medium with antibiotics (penicillin/streptomycin) to a final concentration of C.sub.E100 mg/ml. [0107] This suspension served as stock solution and was stored at 4 C.

Test Procedure:

[0108] A defined period of time before the start of the experiment, cells of a selected culture (for example, one day before the start of the experiment if MCF-10A cells, ATCC CRL-10317, were used; or 5 days in the case of Caco-2 cells, ATCC HTB-34) were seeded on sterile round glass slides (diameter 10 mm) in petri dishes. [0109] After 24 hours under growth conditions, the cells (1 glass slide per batch) were incubated with 10 g/cm.sup.2 of one of the test substances (see Table 1) under growth conditions for a further 24 hours. [0110] The glass slides were then washed once with 50 mM HEPES buffer, pH 7.2, and fixed at room temperature in 0.5%-2.0% paraformaldehyde in 50 mM HEPES buffer, pH 7.2, for 30 min. [0111] Afterwards, the glass slides were again washed 3 in the above HEPES buffer before being incubated protected from light in 1 ml of [0112] a) 100 M lumogallion solution in 50 mM HEPES buffer, pH 7.2, [0113] or [0114] b) 200 M lumogallion solution in 20 mM acetate buffer, pH 4.0, [0115] for either [0116] 2 to 4 hours at 60 C. to 80 C., [0117] or [0118] 12 to 24 hours at room temperature to 37 C. [0119] Prior to embedding in mounting medium (VECTASHIELD Antifade Mounting Medium, Vector Laboratories, USA), the glass slides were briefly dipped in double-distilled water and drained on a clean paper towel. [0120] The embedded samples were attached to the slide in an airtight manner using a viscous clear coat to prevent desiccation. [0121] The individual samples were then assayed for lumogallion labeling using an epifluorescence microscope and various filters (TRITC, FITC, or morin filters, see Table 2) (see FIGS. 1 and 6b).

Example of Labeling of Aluminosilicates in Histosections:

Test Procedure for Dewaxed Samples:

[0122] 1. The tissue samples embedded in paraffin or paraffin substitute (Paraplast) were sectioned on the cryomicrotome to a specimen thickness of 5 m to 25 M and transferred to glass slides. [0123] 2. Afterwards, the sections were washed 3 with PBS. [0124] 3. This was followed by incubation in Neo-Clear (xylene substitute for microscopy, Merck, Germany) for 3 min under gentle shaking. [0125] 4. Then the used Neo-Clear was removed and replaced with fresh Neo-Clear, which in turn remained on the sample under gentle shaking. This incubation lasted 1 min. [0126] 5. Afterwards, the section was subjected to several washing steps: [0127] 3 with PBS [0128] 3 with EtOH.sub.absolut [0129] 3 with 50 mM Hepes, pH 7.2, [0130] 6. Followed by incubation with [0131] a) 100 M lumogallion in 50 mM HEPES buffer, pH 7.2, [0132] or [0133] b) 200 M lumogallion in 20 mM acetate buffer, pH 4.0, [0134] for 12 to 24 hours (overnight) at 37 C. [0135] 7. In order to achieve a longer shelf life of the fluorescence staining, the sections were covered with mounting medium (for example VECTASHIELD Antifade Mounting Medium, Vector Laboratories, USA) and covered with a cover slip. [0136] 8. The embedded sections were protected from desiccation by attaching the cover slip to the slide using a viscous clear coat. [0137] 9. The individual samples were then assayed for lumogallion labeling using a fluorescence microscope and various filters (TRITC, FITC, or morin filters, see Table 2).
Test Procedure for Samples Coated with Paraffin:

[0138] Paraffin sections were prepared as described in the above protocol (Item 1), washed on the slides (Item 5), incubated with lumogallion (Item 6), embedded (Items 7 and 8), and analyzed using epifluorescence microscopy (Item 9). Items 2, 3, and 4 were omitted. FIG. 4 will be used as an example of the application.

Example of Labeling of Aluminosilicates in Mineralogical-Geological Samples:

[0139] 1. The glass slides were cleaned with 70% ethanol to allow adhesion of the liquid blocker super PAP pen (Liquid-Repellent Slide Marker Pen, Science Services, Germany). It was used to draw rectangles on the glass, which after drying were used as tubs for filling with buffer or lumogallion solution. [0140] 2. The rock samples were ground to fine particles (approximately 1 m to 60 m in diameter) (see Table 1) and embedded in paraffin substitute (Paraplast X-Tra Tissue Infiltration/Embedding Medium, McCormick Scientific, PA), cut to a specimen thickness of 14 m to 16 m (similar to that of the tissue samples) on the cryomicrotome before being transferred to the previously prepared glass slides. [0141] 3. The sections floating in the buffer or lumogallion solution were incubated at 37 C. for 12-24 hours. [0142] 4. Afterwards, they were applied to new slides, overlaid with VECTASHIELD Antifade Mounting Medium (Vector Laboratories, USA) and covered with a cover slip. [0143] 5. The edges between the slides and cover slips were sealed with transparent clearcoat. [0144] 6. After the clearcoat dried, the samples were examined using epifluorescence microscopy (see FIG. 6aindividual substances, and FIG. 2mixtures). For this, the various filters described in Table 2 (TRITC, FITC, or morin filters) were used.

TABLE-US-00001 TABLE 1 Table 1: Test substances used (selection) and their manufacturers or distributors. Substance Product lnformation/(Product Number) Activated carbon Activated carbon, pure Merck (102183) Aluminum oxide Aluminum oxide Sigma-Aldrich (342750) Barium sulfate Prepared from Barium chloride dihydrate 99.995% Suprapur Sigma-Aldrich (101716) and Anhydrous sodium sulfate for analysis ACS, ISO, Reag. Ph Eur Merck (106649) Calcium Calcium carbonate, precipitated, analytical carbonate grade Ph.Eur., USP AppliChem (A0774) Feldspar AUT Feldspar, dry Amberger Kaolinwerke (FS900 SF Hirschau) Feldspar USA Feldspar Fortispar K-30 I Minerals Inc. (ULTRA HalloPure) Halloysite Halloysite I Minerals Inc. (ULTRA HalloPure) Clinoptilolite Glock Health, Science and Research (018-01-08-2-1-1) (Example of a natural zeolite whose heavy metal ions were exchanged for calcium according to U.S. Pat No. 8,173,101 B2.) Kaolin Kaolin chamotte Amberger Kaolinwerke (AS45 6.400 Hirschau) Montmorillonite Montmorillonite naturally occurring mineral Alfa Aesar/VWR (42531.22) Silicon Silicon powder, APS 1-5 micron, 99.9% (metals basis) Alfa Aesar/Thermo Fischer (Kandel) (44185) Titanium dioxide Titanium(IV)oxide, anatase Sigma-Aldrich (248576) Zeolite HY Zeolyst International (CBV400) (Example of an artificially synthesized zeolite)

TABLE-US-00002 TABLE 2 Table 2: Fluorescence filters used Filter .sub.Absorption [nm] .sub.Emission [nm] TRITC 546/12 (band pass) 575-640 (band pass) 560 (beam splitter) FITC 450-490 (band pass) 515-565 (band pass) 510 (beam splitter) Morin 433/24 (band pass) >473 (long pass) 465 (beam splitter)

[0145] Using a mounting medium and fluorescence microscopy and various filters (TRITC, FITC, morin), the samples prepared according to the invention can be stably detected and differentiated from a large number of non-aluminosilicates for a period of time ranging from days to several months. In cell biological and histological samples, which were embedded in either paraffin or paraffin substitute (Paraplast or the like), the samples can be labeled without the need for dewaxing using the method according to the invention.

[0146] Compared to the prior art, this constitutes a quick, simple, reliable, and cost-effective method.

[0147] As used in the description and claims, substantially means a deviation of up to 10% of the stated value, if this is physically possible, both downwards and upwards, otherwise only in the reasonable direction; indications in regard to temperatures are thus meant to be read as deviations of 10 C.

[0148] All quantities and proportions, in particular those for delimiting the invention, as far as these do not relate to the specific examples, are to be understood with 10% tolerance. Thus, for example: 11% means: from 9.9% to 12.1%. Percentages of ingredients are by weight unless otherwise specified. For terms such as a solvent, the word a is not to be regarded as a numerical word, but as a pronoun or as an indefinite article, unless the context indicates otherwise.

[0149] The term: Combination or Combinations means, unless otherwise indicated, all types of combinations, starting from two of the relevant constituents, to a multiplicity of such constituents, to all constituents. The term comprising also means consisting of.

[0150] The characteristics and variants specified in the individual embodiments and examples may be freely combined with those of the other examples and embodiments, and may in particular be used to characterize the invention in the claims without necessarily entraining the other details of the respective embodiment or the respective example.

REFERENCES

Non-Patent Literature (Shortened to the Most Important Publications)

Babourina O, Rengel Z.

[0151] Uptake of aluminium into Arabidopsis root cells measured by fluorescent lifetime imaging.

Ann Bot. 2009; 104(1): 189-195.

[0152] doi: 10.1093/aob/mcp098

Caschetto S, Wollast R.

[0153] Dissolved aluminum in interstitial waters of recent marine sediments.

Geochim Cosmochim Acta. 1979; 43: 425-428.

Coons A H, Creech H J, Jones R N.

[0154] Immunological properties of an antibody containing a fluorescent group.

Proc Soc Exp Biol Med. 1941; 47: 200-202.

Coons A H, Creech H J, Jones R N, Berliner E.

[0155] The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody.

J Immunol. 1942; 45: 159-170.

Coons A H, Melvin H, Kaplan M H.

[0156] Localization of antigen in tissue cells: II. Improvements in a method for the detection of antigen by means of fluorescent antibody.

J Exp Med. 1950; 91(1): 1-13.

Cordes T, Maiser A, Steinhauer C, Schermelleh L, Tinnefeld P.

[0157] Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy.

Phys Chem Chem Phys. 2011; 13(14): 6699-6709.

[0158] doi: 10.1039/c0cp01919d

Crompton T R.

[0159] Determination of metals in natural waters, sediments, and soils.
Elsevier, Amsterdam. 2015; 1.sup.st Edition: 318 pages.

Eticha D, Stass A, Horst W J.

[0160] Localization of aluminium in the maize root apex: can morin detect cell wall-bound aluminium?

J Exp Bot. 2005; 56(415): 1351-1357.

[0161] doi:10.1093/jxb/eri136

Florijn R J, Slats J, Tanke H J, Raap A K.

[0162] Analysis of antifading reagents for fluorescence microscopy.

Cytometry. 1995; 19(2): 177-182.

[0163] doi: 10.1002/cyto.990190213

He H-B, Lee H-K, Li S F Y, Hsieh A-K, Chi H, Siow K-S.

[0164] Determination of Trace Levels of Aluminum by Capillary Electrophoresis with Lumogallion Fluorometric Detection.

J Chromatogr Sci. 1997; 35(7): 333-336.

[0165] doi: 10.1093/chromsci/35.7.333

Huff J C, Weston W L, Wanda K D.

[0166] Enhancement of specific immunofluorescent findings with use of a para-phenylenediamine mounting buffer.

J Invest Dermatol. 1982; 78(5): 449-450.

Hydes D J, Liss P S.

[0167] The determination of low concentrations of dissolved aluminum in natural waters.

Analyst. 1976; 101: 922-931.

Ills P, Schlicht M, Pavlovkin J, Lichtscheidl I, Baluska F, Ovecka M.

[0168] Aluminium toxicity in plants: internalization of aluminium into cells of the transition zone in Arabidopsis root apices related to changes in plasma membrane potential, endosomal behaviour, and nitric oxide production.

J Exp Bot. 2006; 57(15): 4201-4213.

[0169] doi: 10.1093/jxb/erl197

Johnson G D, Nogueira Araujo G M.

[0170] A simple method of reducing the fading of immunofluorescence during microscopy.

J Immunol Methods. 1981; 43: 349-350.

Johnson G D, Davidson R S, McNamee K C, Russell G, Goodwin D, Holborow E J.

[0171] Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy.

J Immunol Methods. 1982; 55(2): 231-242.

Jonasson R G.

[0172] Investigation of the reactions of aquatic aluminum through fluorometry using lumogallion.
McMaster University, 1980. [http://hdl.handle.net/11375/18023].

A Thesis Submitted to the Department of Geology in Partial Fulfillment of the Requirements for the Degree Bachelor of Science.

Kataoka T, Iikura H, Nakanishi T M.

[0173] Aluminum distribution and viability of plant root and cultured cells.

Soil Science and Plant Nutrition. 1997; 43: 1003-1007.

[0174] doi: 10.1080/00380768.1997. Ser. No. 11/863,707

Klein J P, Mold M, Mery L, Cottier M, Exley C.

[0175] Aluminum content of human semen: implications for semen quality.

Reprod Toxicol. 2014; 50: 43-48.

[0176] doi: 10.1016/j.reprotox.2014.10.001

Lee B L, Chua L H, Ong H Y, Yang H G, Wu J, Ong C N.

[0177] Determination of serum and urinary aluminum by HPLC with fluorometric detection of Al-lumogallion complex.

Clin Chem. 1996; 42(9): 1405-1411.

Li Z, Xing F, Xing D.

[0178] Characterization of target site of aluminum phytotoxicity in photosynthetic electron transport by fluorescence techniques in tobacco leaves.

Plant Cell Physiol. 2012; 53(7): 1295-1309.

[0179] doi: 10.1093/pcp/pcs076

Longin A, Souchier C, Ffrench M, Bryon P A.

[0180] Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study.

J Histochem Cytochem. 1993; 41(12): 1833-1840.

Mile I, Svensson A, Darabi A, Mold M, Siesj P, Eriksson H.

[0181] Al adjuvants can be tracked in viable cells by lumogallion staining.

J Immunol Methods. 2015; 422: 87-94.

[0182] doi: 10.1016/j.jim.2015.04.008

Ming D W, Dixon J B.

Quantitative Determination of Clinoptilolite in Soils by a Cation-Exchange Capacity Method.

Clays and Clay Minerals. 1987; 35: 463-468.

Mirza A, King A, Troakes C, Exley C.

The Identification of Aluminum in Human Brain Tissue Using Lumogallion and Fluorescence Microscopy.

J Alzheimers Dis. 2016; 54(4): 1333-1338.

doi: 10.3233/JAD-160648

Mirza A, King A, Troakes C, Exley C.

[0183] Aluminium in brain tissue in familial Alzheimer's disease.

J Trace Elem Med Biol. 2017; 40: 30-36.

[0184] doi: 10.1016/j.jtemb.2016.12.001

Mold M, Eriksson H, Siesj P, Darabi A, Shardlow E, Exley C.

[0185] Unequivocal identification of intracellular aluminium adjuvant in a monocytic THP-1 cell line.

Sci Rep. 2014; 4: 6287.

[0186] doi: 10.1038/srep06287

Mold M, Shardlow E, Exley C.

[0187] Insight into the cellular fate and toxicity of aluminium adjuvants used in clinically approved human vaccinations.

Sci Rep. 2016; 6: 31578.

[0188] doi: 10.1038/srep31578

Mumpton F A.

[0189] La roca magica: uses of natural zeolites in agriculture and industry.

Proc Natl Acad Sci USA. 1999; 96(7): 3463-3470.

Nishikawa Y, Hiraki K, Morishige, Shigematsu T.

[0190] Fluorophotometric determination of aluminum and gallium with lumogallion.

Japan Analyst (Bunseki Kagaku). 1967; 16: 692-697.

Nishikawa Y, Hiraki K, Morishige K, Tsuchiyama A, Shigematsu T.

[0191] Fluorometric determination of trace amount of aluminum in sea water.

Japan Analyst (Bunseki Kagaku). 1968; 17: 1092-1097.

Platt J L, Michael A F.

[0192] Retardation of fading and enhancement of intensity of immunofluorescence by p-phenylenediamine.

J Histochem Cytochem. 1983; 31(6): 840-842.

[0193] doi: 10.1177/31.6.6341464

Powell J J.

[0194] Analysis of aluminosilicate particles in biological matrices using histochemistry and X-ray microanalysis.

Analyst. 2002; 127: 842-846.

doi: 10.1039/B110761P

Ramos Jareo M E.

[0195] Dissolution mechanism of montmorillonite in synthetic lung fluids: effect of organic ligands and biodurability.
Doctoral dissertation.

Granada: Universidad de Granada, 2013. 294 p.

[0196] Retrieved from http://digibug.ugr.es/handle/10481/29552#.WYLoR7mQqHs.

Ren J L, Zhang J, Luo J Q, Pei X K, Jiang Z X.

[0197] Improved fluorimetric determination of dissolved aluminium by micelle-enhanced lumogallion complex in natural waters.

Analyst. 2001; 126(5): 698-702.

[0198] doi: 10.1039/b007593k

Shigematsu T, Nishikawa Y, Hiraki K, Nagano N.

[0199] Fluorometric determination of trace amount of aluminum in natural water by lumogallion method. Masking of ferric iron with o-phenanthroline.

Japan Analyst (Bunseki Kagaku). 1970; 19(4): 551-554.

Sivaguru M, Liu J, Kochian L V.

[0200] Targeted expression of SbMATE in the root distal transition zone is responsible for sorghum aluminum resistance.

Plant J. 2013; 76(2): 297-307.

[0201] doi: 10.1111/tpj.12290

Sposito G.

[0202] The environmental chemistry of aluminum.
CRC Press, Boca Raton. 1995; 2.sup.nd Edition: 480 pages.

Sutheimer S H, Maurice P A, Zhou Q.

[0203] Dissolution of well and poorly crystallized kaolinites; Al speciation and effects of surface characteristics.

American Mineralogist. 1999; 84(4): 620-628.

[0204] doi: 10.2138/am-1999-0415

Tibbett M, Carter D O.

Soil Analysis in Forensic Taphonomy: Chemical and Biological Effects of Buried Human Remains.

[0205] CRC Press. Feb. 27, 2008; 364 pages.

Uchiumi A, Takatsu A, Teraki Y.

[0206] Sensitive detection of trace aluminium in biological tissues by confocal laser scanning microscopy after staining with lumogallion.

Analyst. 1998; 123(4): 759-762.

Valnes K, Brandtzaeg P.

[0207] Retardation of immunofluorescence fading during microscopy.

J Histochem Cytochem. 1985; 33(8): 755-761.

[0208] doi:10.1177/33.8.3926864

Yamada E, Hiwada T, Inaba T, Tokukura M, Fuse Y.

[0209] Speciation of aluminum in soil extracts using cation and anion exchangers followed by a flow-injection system with fluorescence detection using lumogallion.

Anal Sci. 2002; 18(7): 785-791.

Zhou Y, Yokel R A.

[0210] The chemical species of aluminum influences its paracellular flux across and uptake into Caco-2 cells, a model of gastrointestinal absorption.

Toxicol Sci. 2005; 87(1): 15-26.

[0211] doi: 10.1093/toxsci/kfi216

Patent Literature

[0212] Anti-counterfeit penetrant atomic stamp pad ink and its preparation method, 1996 [0213] CN 96104762 [0214] Anti-fake fluorescent pressure-sensitive carbon paper generating invisible duplication handwriting and making method thereof, 1996 [0215] CN 96100340 [0216] Anti-fake fluorescent pressure-sensitive carbon paper, 1996 [0217] CN 96100339 [0218] Changed condition indicator, 2001 [0219] U.S. Ser. No. 11/375,307 [0220] Chelate reagent and measuring aluminum and measuring method, 2000 [0221] U.S. Ser. No. 10/380,242 [0222] Detector used for investigating water consists of an active ingredient coating formed on a carrier, 2000 [0223] DE2000160845 [0224] Diagnostic test for elemental imbalances, 2002 [0225] PCT/US2003/012911 and EP20030719936 [0226] Dye lasers, 1978 [0227] U.S. Ser. No. 05/963,157 [0228] Fiber optic moisture sensor, 1992 [0229] U.S. Ser. No. 07/914,795 and U.S. Ser. No. 08/201,820 [0230] Fluorescent anti-fake mark and its making method, 1994 [0231] CN 94105055 [0232] Fluorescent probe for the detection of corrosion, 2005 [0233] PCT/G B2006/001626 [0234] Method for analyzing trace quantity of Aluminum, 2003 [0235] JP20020104985 20020408 [0236] Method for determination of aluminum, 2001 [0237] U.S. Ser. No. 10/498,060 [0238] Methods of monitoring rinsing solutions and related systems and reagents, 2004 [0239] U.S. Ser. No. 10/952,510 [0240] Method of producing crosslinkable silyl group-containing polyoxyalkylene polymers, 2000 [0241] U.S. Ser. No. 09/832,344 and EP20010109302 [0242] Organic electroluminescence device, 2000 [0243] U.S. Ser. No. 09/915,452 [0244] Organic LED element, 1999 [0245] JP19980083987 19980330 [0246] pH-sensitive microparticles with matrix-dispersed active agent, 2011 [0247] U.S. Ser. No. 13/542,155 [0248] Photodeposition method for fabricating a three-dimensional, patterned polymer microstructure, 1995 [0249] U.S. Ser. No. 08/519,062 [0250] Semiconductor device and adhesive sheet, 2009 [0251] CN 201010135730 [0252] Semiconductor wafer cleaning agent and cleaning method, 2000 [0253] U.S. Ser. No. 10/203,659 [0254] Synergistic combinations of chromate-free corrosion inhibitors, 2005 [0255] PCT/US2006/007305 and U.S. Ser. No. 11/817,659 [0256] Verfahren zur Herstellung vernetzbarer Silylgruppen enthaltender Polyoxyalkylenpolymere, 2000 [0257] DE2001625268

[0258] The content of the English language references, especially of the patent literature, is incorporated herein by reference for the jurisdictions in which this is possible.