WO2012096842A1 - Hematoxylin staining method - Google Patents
Hematoxylin staining method Download PDFInfo
- Publication number
- WO2012096842A1 WO2012096842A1 PCT/US2012/020483 US2012020483W WO2012096842A1 WO 2012096842 A1 WO2012096842 A1 WO 2012096842A1 US 2012020483 W US2012020483 W US 2012020483W WO 2012096842 A1 WO2012096842 A1 WO 2012096842A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hematein
- mordant
- solution
- biological sample
- solutions
- Prior art date
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- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 title claims abstract description 134
- 238000007447 staining method Methods 0.000 title description 3
- HLUCICHZHWJHLL-UHFFFAOYSA-N hematein Chemical compound C12=CC=C(O)C(O)=C2OCC2(O)C1=C1C=C(O)C(=O)C=C1C2 HLUCICHZHWJHLL-UHFFFAOYSA-N 0.000 claims abstract description 156
- 239000012472 biological sample Substances 0.000 claims abstract description 55
- 238000000034 method Methods 0.000 claims abstract description 51
- 238000010186 staining Methods 0.000 claims abstract description 49
- 230000008569 process Effects 0.000 claims abstract description 28
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 54
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- 239000000758 substrate Substances 0.000 claims description 18
- 239000007800 oxidant agent Substances 0.000 claims description 16
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 15
- 239000002904 solvent Substances 0.000 claims description 15
- 239000012530 fluid Substances 0.000 claims description 9
- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 claims description 6
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- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
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- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 239000001017 thiazole dye Substances 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 229950003937 tolonium Drugs 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
- DKBXPLYSDKSFEQ-UHFFFAOYSA-L turquoise gll Chemical compound [Na+].[Na+].[Cu+2].N1=C(N=C2[N-]3)[C]4C(S(=O)(=O)[O-])=CC=CC4=C1N=C([N-]1)C4=CC=CC(S([O-])(=O)=O)=C4C1=NC(C=1C4=CC=CC=1)=NC4=NC3=C1[C]2C=CC=C1 DKBXPLYSDKSFEQ-UHFFFAOYSA-L 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- LSGOVYNHVSXFFJ-UHFFFAOYSA-N vanadate(3-) Chemical compound [O-][V]([O-])([O-])=O LSGOVYNHVSXFFJ-UHFFFAOYSA-N 0.000 description 1
- AODQPPLFAXTBJS-UHFFFAOYSA-M victoria blue 4R Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC(=CC=1)N(C)C)=C(C=C1)C2=CC=CC=C2C1=[N+](C)C1=CC=CC=C1 AODQPPLFAXTBJS-UHFFFAOYSA-M 0.000 description 1
- JEVGKYBUANQAKG-UHFFFAOYSA-N victoria blue R Chemical compound [Cl-].C12=CC=CC=C2C(=[NH+]CC)C=CC1=C(C=1C=CC(=CC=1)N(C)C)C1=CC=C(N(C)C)C=C1 JEVGKYBUANQAKG-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
- G01N1/312—Apparatus therefor for samples mounted on planar substrates
Definitions
- the present invention relates to processes for staining biological samples, and in particular to automated processes for staining biological sample with hematoxylin stains.
- Hematoxylin and Eosin (H&E) staining and Papanicolaou (PAP) staining rely on the dye hematoxylin to stain cytological and tissue samples.
- H&E staining and PAP staining rely on the dye hematoxylin to stain cytological and tissue samples.
- hematoxylin staining of cell nuclei is used by pathologists to detect the presence of malignant and/or metastatic cells in a tumor biopsy sample.
- Hematoxylin is a naturally-occurring compound found in the red heartwood of trees of the genus Hematoxylon. Hematoxylin itself is colorless in aqueous solution and is not the active ingredient that stains tissue components. Rather, an oxidation product of hematoxylin, hematein, becomes the active staining component of a hematoxylin dye solution, particularly upon complexation with a mordant. Hematein is produced naturally through exposure to air and sunlight. The natural process is termed "ripening," and can take 3 or more months to provide a solution suitable for staining cells.
- Standard hematein staining procedures utilized a premixed stock containing both the hematoxylin/hematein and a mordant. Precipitates form in these premixed stocks. This is not generally a problem for manual staining procedures, where slides are treated with the hematoxylin staining solution in a container, such as a glass container.
- precipitates are a problem for automated staining systems where the precipitate can foul or clog delivery lines and make cleaning or purging of the delivery lines difficult.
- hematoxylin stain stocks containing mordant are often allowed to ripen for an extended period of time, allowing developing of hematein-mordant complexes. While this process may allow for good staining results, it also results in formation of the undesirable precipitate. Precipitation is exacerbated by contact with metal. This is especially problematic for automated systems which contain metal parts such as nozzles and sprayheads with very small diameter openings which can be clogged by precipitates.
- the present invention relates to processes for staining biological samples, and in particular to automated processes for staining biological sample with hematoxylin stains.
- the present invention provides processes for staining a biological sample comprising cells, comprising: providing separate hematein and mordant solutions; preparing a fresh hematein-mordant solution by mixing the separate hematein and mordant solutions; and contacting the biological sample with the fresh hematein-mordant solution under conditions such that structures in the cells of the biological sample are stained.
- the hematein and mordant solutions are mixed for a time period selected from the group consisting of less than about 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 10 minutes, 5 minutes, 1 minute, 30 seconds and 20 seconds prior to application to the biological sample.
- the hematein and mordant solutions are applied to the sample as separate solutions and mixed on the sample to provide a fresh hematein-mordant solution.
- the ratio of the separate hematein and mordant solutions is varied to adjust at least one property of the hematein-mordant solution.
- the at least one property is stain intensity.
- hematein solution is a half-oxidized hematoxylin solution.
- the half-oxidized hematoxylin solution comprises an oxidizing agent in an amount sufficient to oxidize about 50 percent of the hematein in the hematein solution.
- the mordant solution comprises aluminum sulfate.
- the sample is mounted on a slide.
- the hematein and mordant solutions comprise a low volatility solvent.
- the low volatility solvent is selected from the group consisting of glycerol, polyethylene glycol and propylene glycol.
- the present invention provides processes for staining a biological sample comprising cells, comprising: providing separate hematein and mordant solutions; mixing the separate hematein and mordant solutions immediately before application to the biological sample to provide a hematein -mordant solution; and applying the hematein mordant solution to the biological sample under conditions such that structures in the cells of the biological sample are stained.
- the present invention provides processes for staining a biological sample comprising cells, comprising: providing separate hematein and mordant solutions; mixing the separate hematein and mordant solutions for a time period of less than about 30 minutes prior to application to the biological sample to provide a hematein-mordant solution; and applying the hematem mordant solution to the biological sample under conditions such that structures in the cells of the biological sample are stained.
- the hematein solution is a half- oxidized hematein solution.
- the mordant solution comprises aluminum sulfate.
- the tissue is mounted on a slide.
- the hematein and mordant solutions comprise a low volatility solvent.
- the low volatility solvent is selected from the group consisting of glycerol, polyethylene glycol and propylene glycol.
- the present invention provides systems for staining a biological sample mounted on a substrate comprising: a first container containing a hematein solution and a second container containing a mordant solution, the first and second containers fluidically connected to a mixing receptacle so that the hematein solution and the mordant solution can be combined to provide a hematein-mordant solution; a substrate holder in fluid communication with the mixing receptacle, so that the hematein-mordant solution can be applied to the biological sample mounted on a substrate when the substrate occupies the substrate holder.
- the mixing receptacle is a tube that is fluidically connected to the first and second containers.
- the system is automated.
- the systems further comprise additional containers containing one or more tissue staining or labeling agents.
- the present invention provides systems for staining a biological sample mounted on a substrate comprising: a first container containing a hematein solution and a second container containing a mordant solution, the first and second containers fluidically connected to dispensers so that the hematein solution and the mordant solution can be applied to the sample mounted on the substrate; a substrate holder in fluid communication with the dispenser, so that the solutions can be applied to the biological sample mounted on a substrate when the substrate occupies the substrate holder.
- a host compound refers to one or more host compounds, such as 2 or more host compounds, 3 or more host compounds, or even 4 or more host compounds.
- biological sample refers to any sample that is obtained from or otherwise derived from a biological entity such as an animal, for example, a sample obtained from a human or a veterinary animal such as a dog, cat, horse or cow.
- biological samples include cytology samples, tissue samples and biological fluids.
- Non-limiting particular examples of biological samples include blood, urine, pre-ejaculate, nipple aspirates, semen, milk, sputum, mucus, pleural fluid, pelvic fluid, sinovial fluid, ascites fluid, body cavity washes, eye brushings, skin scrapings, a buccal swab, a vaginal swab, a pap smear, a rectal swab, an aspirate, a needle biopsy, a section of tissue obtained for example by surgery or autopsy, plasma, serum, spinal fluid, lymph fluid, sweat, tears, saliva, tumors, organs and samples obtained from in vitro cell or tissue cultures.
- the sample will be a biopsy sample that has been fixed, processed to remove water and embedded in paraffin or another suitable waxy substance for cutting into tissue sections.
- Biological samples can be mounted on substrates such as microscope slides for treatment and/or examination.
- hematein solution refers both to compositions formed by dissolving hematein (the oxidation product of hematoxylin) directly into a solvent and to compositions formed by dissolving hematoxylin in a solvent and allowing or promoting oxidation of the hematoxylin to hematein.
- fresh hematein-mordant solution refers to a solution prepared by mixing a hematein solution and a mordant solution either immediately prior to application to a biological sample, for example, by mixing for a time period selected from the group consisting of less than about 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 10 minutes, 5 minutes, 1 minute, 30 seconds and 20 seconds prior to application to the biological sample, or where the hematein and mordant solutions are applied separately to the biological sample and mixed on the sample, for example, by agitation with jets of gas or air.
- oxidant refers to an atom or molecule having a greater reduction potential than a second molecule, for example, a greater reduction potential than hematoxylin such that it will react with and oxidize hematoxylin to hematein.
- Oxidants include naturally occurring molecular oxygen in the atmosphere that diffuses to and oxidizes hematoxylin and a "chemical oxidant" that is actively combined with hematoxylin (typically in solution) to convert at least a portion of the hematoxylin to hematein.
- Half-oxidized hematoxylin solutions are solutions in which the oxidant is included in an amount which oxidizes approximately one half of the available hematoxylin, as described by Gill, Acta Cytologica, 18(4):300-11 (1974), incorporated herein by reference in its entirety.
- useful chemical oxidants include one or more of an iodate salt (such as sodium iodate and potassium iodate), mercuric oxide, a permanganate salt (such as potassium permanganate), a periodate salt (such as sodium periodate and potassium periodate), and a peroxide (such as hydrogen peroxide).
- the chemical oxidant comprises sodium iodate.
- mordant refers to an ionic metal species with which a dye (such as hematein) can form a complex (such as a cationic complex) that serves to bind the dye (such as hematein) to particular cellular components such as nuclear DNA, myelin, elastic and collagen fibers, muscle striations and mitochondria.
- a dye such as hematein
- a complex such as a cationic complex
- mordants include aluminum (for example, in the form of an alum such as aluminum sulfate, aluminum potassium sulfate or aluminum ammonium sulfate), iron, tungsten, zirconium, bismuth, molybdenum (phosphomolybdic acid or molybdic acid), vanadium (vanadate).
- the present invention relates to processes for staining biological samples, and in particular to automated processes for staining biological sample with hematoxylin stains.
- the present invention provides processes for staining a biological sample where separate hematein and mordant solutions are mixed immediately before application to the biological sample. Surprisingly, it has been found that consistent staining results can be obtained when the hematein and mordant solutions are combined immediately before application as opposed to the use of solutions where the hematein and mordant have been present in the staining solution for an extended period of time.
- the hematein and mordant solutions are mixed for a period of time from about less than 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 10 minutes, 5 minutes, 1 minute, 30 seconds or 20 seconds prior to application to the biological sample. In some preferred embodiments, the hematein and mordant solutions are mixed for less than about 1 minute, 30 seconds or 20 seconds prior to application to the biological sample.
- the system comprises a separate reservoirs or containers containing separate hematein and mordant solutions.
- the systems further comprise a dispensing system that delivers the solutions to the biological sample, preferably a biological sample mounted on a slide.
- the hematein and mordant solutions are combined prior to application to the biological sample.
- the container or mordant solution and container of hematein solution are pressurized and fluidically connected to a mixing receptacle.
- the mixing receptacle can be any container capable of holding or transporting the mixed solution such as a rigid or flexible tube.
- the mixing receptacle is a tube that is fluidically connected to the dispenser.
- the hematein and mordant solutions are fluidically connected to a T-fitting via tubing. The output from the T fitting is in turn fluidically connected to the dispenser.
- the mordant and hematein solutions are fed into the T fitting and mixing of the solutions occurs in the tube leading out of the T fitting.
- the separate hematein and mordant solution are separately dispensed onto the biologically sample.
- the solutions can be allowed to mix by diffusion on the sample or mechanically mixed, for example, by agitation with a pipette.
- the amount of chemical oxidant utilized in some embodiments of the composition can be sufficient to completely (such as substantially quantitatively) oxidize the hematoxylin to hematein, or sufficient only to partially oxidize the hematoxylin to hematein. In particular embodiments, more than half of the hematoxylin is oxidized to hematein by the chemical oxidant, and in others, less than half of the hematoxylin is oxidized to hematein by the chemical oxidant.
- the hematoxylin can be oxidized to hematein by the chemical oxidant, but more typically, between about 10% and about 50% of the hematoxylin is oxidized to hematein by the chemical oxidant.
- the molar ratio of hematoxylin to oxidant used in the composition is between 6:1 and 1 : 1. It should be understood that although the chemical oxidant is considered part of the composition, it is converted to its reduction products upon reaction with the hematoxylin, which reduction products will remain in the composition.
- the mordant of the composition can be any mordant such as one or more of an aluminum mordant, an iron mordant, a bismuth mordant, a copper mordant, a molybdenum mordant, a vanadium mordant, and a zirconium mordant.
- the mordant comprises an alum, and in more particular embodiments, the mordant comprises aluminum sulfate.
- the mordant can be present in the composition at a concentration greater than the concentration of the hematein in the composition (determinable by refractometry, thin-layer chromatography or spectroscopy), or it can be present in the composition at a concentration less than the concentration of the hematein in the composition.
- the molar ratio of hematoxylin to mordant in the composition is between 2:1 and 1 : 100, and in particular embodiments, the molar ratio of hematoxylin to mordant in the composition is between 1 :5 and 1 :20.
- the hematein solutions of the present invention comprise hematoxylin (which is oxidized to hematein), a buffer system, an aqueous solvent, and a chemical oxidant.
- the buffer is used in a concentration sufficient to control the pH near a pH between 1 and 4, such as a pH near 2.4-2.8.
- the buffer system comprises phthalic acid and potassium hydrogen phthalate.
- the solvent used to dissolve the hematoxylin comprises an aqueous composition such as composition including water and a low volatility solvent such a polyol.
- Useful polyols include glycerol, ethylene glycol and propylene glycol.
- the mordant solutions of the present invention comprise a mordant, a buffer system, and an aqueous solvent.
- the buffer is used in a concentration sufficient to control the pH near a pH between 1 and 4, such as a pH near 2.4-2.8.
- the buffer system comprises phthalic acid and potassium hydrogen phthalate.
- the solvent used to dissolve the mordant comprises an aqueous composition such as composition including water and a low volatility solvent such a polyol.
- Useful polyols include glycerol, ethylene glycol and propylene glycol.
- Ventana Medical Systems, Inc. is the assignee of a number of United States patents disclosing systems and methods for performing automated analyses, including U.S. Pat. Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901 and 6,943,029, and U.S. published application Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference. These systems may be adapted to be compatible with the present invention.
- the automated slide processing system that is described in the aforementioned applications is a high- volume slide processing system that shuttles trays holding a plurality of slides in substantially horizontal positions (to minimize cross- contamination) between workstations that perform various slide processing operations on the slides.
- Fresh reagents can be applied to each slide during processing, and cross-contamination of slides with reagents can be substantially eliminated because the slides are treated separately in spaced-apart fashion in the tray.
- the system includes a radiant heater, a combined de- paraffinizer/stainer/solvent exchanger workstation, a convection oven and a coverslipper.
- a tray of slides bearing paraffin-embedded tissue samples can be heated under the radiant heater of the system to spread the paraffin in the samples for easier removal and also to adhere the samples to the slides.
- the tray can then be transported to the multifunctional de-paraffinizer/stainer/solvent exchanger workstation, where slides can be de-paraffinized, stained, and solvent exchanged.
- a tray of stained slides that is ready for coverslipping can then be shuttled to the coverslipper of the system where coverslips are added to the slides. Once the slides are coverslipped, the tray can then be transported to the convection oven to cure the coverslips on the stained slides.
- the high volume stainer just described is
- an exemplary H & E protocol is: a baking step to adhere the samples to the slides, a de-paraffmization step to remove paraffin from paraffin- embedded samples, a hematoxylin staining step (that can utilize the disclosed hematoxylin compositions), a bluing step that raises the pH and turns the
- hematoxylin blue to provide better contrast with the eosin added downstream
- an eosin staining step a differentiation step that is used to remove excess eosin and turn the eosin various shades of red to pink
- a dehydration step to remove water from the sample using 100% ethanol
- a step in which the slides are exposed to an elevated temperature and air flow to remove the ethanol a coverslipping step in which limonene is dispensed to the sample, and a curing step.
- alum mordanted hematoxylin histological stains to which a host compound and/or an antioxidant can be added to improve stability
- a host compound and/or an antioxidant can be added to improve stability
- hematoxylins include Bensley's, Cook's and Faure's.
- a molybdenum-mordanted hematoxylin is Held's.
- Vanadium-mordanted hematoxylins include Hedenhain's, and Smith's.
- a zirconium-mordanted hematoxylin is McNulty & Smith's.
- Formulas and methods of making and using such mordanted hematoxylin solutions can be found, for example, in the StainsFile (an internet resource for histotechnologists maintained by Bryan Llewellyn); Kiernan, "Histological and Histochemical methods: Theory and Practice," 3rd Ed.
- the systems and methods further comprise staining of biological samples with additional stains, such as counterstains.
- additional stains such as counterstains.
- contacting the sample with a counterstain comprises contacting the sample with one or more of eosin Y, orange G, light green SF yellowish, Bismark Brown, fast green FCF, OA-6, EA25, EA36, EA50 and EA65.
- StainsFile an internet resource for histotechnologists maintained by Bryan Llewellyn
- Kiernan "Histological and Histochemical methods: Theory and Practice,” 3rd Ed. Butterworth Heinemann, Oxford, UK; and in Horobin and Kiernan, "Conn's biological stains: a handbook of dyes, stains and fluorochromes for us in biology and medicine," 10th ed., Oxford: BIOS, ISBN 1859960995, 2002.
- other biological stains a handbook of dyes, stains and fluorochromes for us in biology and medicine
- contacting the sample with the hematoxylin composition comprises a progressive hematoxylin staining protocol. In other embodiments, contacting the sample with the hematoxylin composition comprises a regressive hematoxylin staining protocol.
- the method can be automated, and can be performed on a biological sample that is supported on a substrate such as a microscope slide. In particular embodiments, the method is used to stain a tissue section or a cytology sample mounted on a microscope slide. In particular embodiments further including a counterstaining step, the method can be an H&E staining method or a PAP staining method, and more particularly an automated H&E or PAP staining method.
- dyes such as acridine dyes, anthraquinone dyes, arylmethane dyes, azo dyes, diazonium dyes, nitro dyes, phthalocyanine dyes, quinine imine dyes, tetrazolium dyes, thiazole dyes and xanthene dyes.
- dyes useful for histological staining include acetyl yellow, acid black 1, acid blue 22, acid blue 93, acid fuchsin, acid green, acid green 1, acid green 5, acid magenta, acid orange 10, acid red 4, acid red 26, acid red 29, acid red 44, acid red 51, acid red 66, acid red 73, acid red 87, acid red 91, acid red 92, acid red 94, acid red 101, acid red 103, acid roseine, acid rubin, acid violet 19, acid yellow 1, acid yellow 9, acid yellow 23, acid yellow 24, acid yellow 36, acid yellow 73, acid yellow S, acid yellow T, acridine orange, acriflavine, alcian blue, alcian yellow, alcohol soluble eosin, alizarin, alizarin blue, alizarin blue 2RC, alizarin carmine, alizarin cyanin BBS, alizarol cyanin R, alizarin red S, alizarin purpurin, aluminon, amido black 10B, amidonaphthol
- Sodium iodate 0.12 g For solutions A and B, propylene glycol is heated on a hotplate at 60°C. The phthalic acid is added and the composition is heated until the solid is dissolved. The heat is turned off and a solution of the potassium hydrogen phthalate in the DI H 2 0 is added to the propylene glycol solution while stirring. The potassium aluminum sulfate is added to solution A as a solid. Solution A is then stirred overnight and then filtered using 25 micron fluted filter paper. The hematoxylin is added to solution B and the composition is stirred for approximately 15 minutes. The solid sodium iodate is then added and the solution is stirred overnight and then filtered using 25 micron fluted filter paper.
- the following protocol was used on a thick film (open) box.
- the slides are manually deparaffinized, lightly blotted and placed in the box.
- 400 ⁇ , of 2X Solution A is dispensed onto the tissue, followed immediately by 400 ⁇ ⁇ of 2X Solution B.
- the slide is then incubated for 3 minutes.
- the slides are then rinsed two times for 10 seconds with 800 1% aqueous Merpol.
- the slides are then treated with 800 ⁇ 95% propylene glycol for 10 seconds.
- the slides are next rinsed three times with 800 ⁇ , transfer fluid for 10 seconds each.
- a Coverslip is then applied on a Sakkura TissueTek instrument.
- Solution A 0.75 M phthalate buffered KA1(S0 4 ) 2 .
- KA1(S0 4 ) 2 0.75 M phthalate buffered KA1(S0 4 ) 2 .
- phthalic acid 8.97 g phthalic acid is added and the mixture stirred at 60°C until the solid dissolves.
- Potassium hydrogen phthalate solution (4.3 g in 750 mL DI H 2 0) is added with stirring and the solution is removed from the heat. 12.0 g
- Solution B 0.75 M phthalate buffered hematein.
- propylene glycol 8.97 g phthalic acid is added and the mixture stirred at 60°C until the solid dissolves.
- Potassium hydrogen phthalate solution (4.3 g in 750 mL DI H 2 0) is added with stirring and the solution is removed from the heat. 12.0 g Hematoxylin is added with stirring. After 15-20 minutes, 1.2 g sodium iodate is added and the mixture stirred overnight. The solution is then filtered. The pH is 2.71.
- a and B solutions were put into bottles on a breadboard and the outputs from the bottles were connected by a T fitting to allow premixing of the two separate solutions.
- a slide (3X3 manually deparaffinized) was placed in an open thin film staining box and the tissue covered with 800 ⁇ ⁇ of 50/50 propylene glycol/H20 mixture to prevent drying of the tissue.
- the premix apparatus described above was pressurized and 2-3 mL of the solutions were dispensed into a tube and transferred to the open thin film box.
- the 50/50 solution was air-knived from the tissue sample and 800 of the premixed Solution A and B was dispensed on top of the slide.
- the slide was incubated for three minutes and then the remaining steps from the protocol in Example 2 were carried out. This procedure was repeated on additional slides with a 3 minute repeat treatment with the stain mix (i.e., two 3 minute incubations) with a six minute incubation. The remaining steps from the protocol in Example 2 were then carried out.
- the two part staining solutions can be used by mixing different volumes of each part, provided the stoichiometry of the component chemicals (aluminum salt/hematein) are maintained via adjustment of the appropriate solution
- hematein-containing solution in which the concentration of the hematein is reduced, but the buffer system is maintained at 0.075 M, is exemplified by the following: Propylene glycol (31.25 mL) was heated to 60°C, and phthalic acid (1.12 g) was added with stirring until dissolved. Remove the mixture from the heat, and add a solution of potassium hydrogen phthalate (0.54 g) in 93.75 mL DI H 2 0 to it. Hematoxylin (1.0 g) was then added with stirring, and after dissolution, sodium iodate (0.1 g) was added and the mixture stirred overnight. After filtration, the mixture was ready for use. (Solution B2).
- Propylene glycol (31.25 mL) was heated to 60°C, and phthalic acid (0.56 g) was added with stirring until dissolved. Remove the mixture from the heat, and add a solution of potassium hydrogen phthalate (0.27 g) in 93.75 mL DI 3 ⁇ 40 to it.
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Abstract
Description
Claims
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
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BR112013017366A BR112013017366A2 (en) | 2011-01-10 | 2012-01-06 | automated process for staining a biological sample comprising cells and system for staining a biological sample mounted on a substrate |
KR1020137017397A KR20140002694A (en) | 2011-01-10 | 2012-01-06 | Hematoxylin staining method |
US13/977,902 US20130302852A1 (en) | 2011-01-10 | 2012-01-06 | Hematoxylin Staining Method |
AU2012205849A AU2012205849A1 (en) | 2011-01-10 | 2012-01-06 | Hematoxylin staining method |
CA2822451A CA2822451A1 (en) | 2011-01-10 | 2012-01-06 | System and method of hematoxylin staining |
EP12701289.6A EP2663852A1 (en) | 2011-01-10 | 2012-01-06 | Hematoxylin staining method |
JP2013548576A JP2014505248A (en) | 2011-01-10 | 2012-01-06 | Hematoxylin staining method |
CN201280012535XA CN103415762A (en) | 2011-01-10 | 2012-01-06 | Hematoxylin staining method |
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EP (1) | EP2663852A1 (en) |
JP (1) | JP2014505248A (en) |
KR (1) | KR20140002694A (en) |
CN (1) | CN103415762A (en) |
AU (1) | AU2012205849A1 (en) |
BR (1) | BR112013017366A2 (en) |
CA (1) | CA2822451A1 (en) |
WO (1) | WO2012096842A1 (en) |
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Also Published As
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KR20140002694A (en) | 2014-01-08 |
US20130302852A1 (en) | 2013-11-14 |
CN103415762A (en) | 2013-11-27 |
EP2663852A1 (en) | 2013-11-20 |
AU2012205849A1 (en) | 2013-07-04 |
CA2822451A1 (en) | 2012-07-19 |
JP2014505248A (en) | 2014-02-27 |
BR112013017366A2 (en) | 2016-10-04 |
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