Patent US3997656A successfully refreshed from Google Patents.
US3997656A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Tissue staining method and composition
Assignee: Applied Bioscience
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Filing: 1975-07-15
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Granted: 1976-12-14
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Expires: 1995-07-15
Abstract
Abstract A superior and extremely rapid histological staining method is described in which frozen tissue is briefly immersed in an aqueous solution comprising trichloroacetic acid, zinc chloride and formaldehyde and then in a second solution comprising hematoxylin. The tissue can be further counterstained if desired. Also disclosed are improved fixatives useful in this and other histological techniques.
Inventors
- 👤 Paul T. Wertlake
- 👤 James S. Harrison
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 08:51
CPC Classifications
G; G01; G01N; G01N1/00; G01N1/28; G01N1/30; A; A01; A01N; A01N1/00; A01N1/10; A01N1/12; A01N1/128; G01N2001/305
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
108 words
A method of staining tissue for microscopic examination which comprises (a) immersing frozen tissue in an aqueous solution comprising from about 1 to about 50% wt/vol. of a mixture of trichloroacetic acid, zinc chloride and formaldehyde wherein said mixture contains from about 5% to about 85%, by weight of said mixture, of trichloroacetic acid, from about 4% to about 30%, by weight of said mixture, of zinc chloride and from about 3% to about 70%, of by weight of said mixture, of formaldehyde, and (b) thereafter immersing the tissue at least once in an aqueous solution comprising 0.1 to 2% hematoxylin until staining of said tissue is complete.
Claim 2
Independent Claim
108 words
A method of staining tissue for microscopic examination which comprises (a) immersing frozen tissue in an aqueous solution comprising from about 1 to about 50% wt/vol. of a mixture of trichloroacetic acid, zinc chloride and formaldehyde wherein said mixture contains from about 5% to about 85%, by weight of said mixture, of trichloroacetic acid, from about 4% to about 30%, by weight of said mixture, of zinc chloride and from about 3% to about 70%, of by weight of said mixture, of formaldehyde, and (b) thereafter immersing the tissue at least once in an aqueous solution comprising 0.1 to 2% hematoxylin until staining of said tissue is complete.
Claim 3
Independent Claim
110 words
A method of staining tissue for microscopic examination which comprises (a) immersing frozen tissue in an 8 to 20% wt/vol solution of a mixture of trichloroacetic acid, zinc chloride and formaldehyde wherein the weight ratio of zinc chloride to trichloroacetic acid is from about 2:1 to about 4:1 and the weight ratio of formaldehyde to trichloroacetic acid is from about 8:1 to about 10:1, in an aqueous lower alkanol wherein the volume ratio of alkanol to total water is from about 1:1 to about 1:3, and (b) thereafter immersing the tissue at least once in an aqueous solution comprising 0.1 to 2% hematoxylin until staining of said tissue is complete.
Claim 4
Independent Claim
110 words
A method of staining tissue for microscopic examination which comprises (a) immersing frozen tissue in an 8 to 20% wt/vol solution of a mixture of trichloroacetic acid, zinc chloride and formaldehyde wherein the weight ratio of zinc chloride to trichloroacetic acid is from about 2:1 to about 4:1 and the weight ratio of formaldehyde to trichloroacetic acid is from about 8:1 to about 10:1, in an aqueous lower alkanol wherein the volume ratio of alkanol to total water is from about 1:1 to about 1:3, and (b) thereafter immersing the tissue at least once in an aqueous solution comprising 0.1 to 2% hematoxylin until staining of said tissue is complete.
Claim 5
Dependent Claim
77 words
The method of claim 2 wherein solution (a) is a 9 to 11% solution wt/vol of a mixture of trichloroacetic acid, zinc chloride and formaldehyde wherein the weight ratio of zinc chloride to trichloroacetic acid is from about 3:1 to about 3.5:1 and the weight ratio of formaldehyde to trichloroacetic acid is from about 8:1 to about 10:1, in aqueous methanol wherein the volume ratio of methanol to total water is from about 1:1.1 to about 1:1.5.
Claim 6
Dependent Claim
12 words
The method of claim 2 wherein said hematoxylin is complexed with alum.
Claim 7
Dependent Claim
32 words
The method of claim 2 wherein the immersion of said tissue in said solution of hematoxylin is preceded by immersion of said tissue in a solution of lithium carbonate at least once.
Claim 8
Dependent Claim
17 words
The method of claim 2 wherein said tissue is counterstained after immersion in said solution of hematoxylin.
Claim 9
Dependent Claim
11 words
The method of claim 6 wherein said counterstain is a eosion.
Claim 10
Dependent Claim
11 words
The method of claim 6 wherein said counterstain is a fuchsin.
Claim 11
Dependent Claim
67 words
A histological fixative composition comprising a solution of (a) a mixture of from about 5% to about 85%, by weight, of trichloroacetic acid, from about 4% to about 30%, by weight, of zinc chloride, and from about 3% to about 70%, by weight, of 100% formaldehyde, in (b) an aqueous medium, the concentration of said mixture in said solution being from about 1% to about 50%, wt/vol.
Claim 12
Dependent Claim
67 words
A histological fixative composition comprising a solution of (a) a mixture of from about 5% to about 85%, by weight, of trichloroacetic acid, from about 4% to about 30%, by weight, of zinc chloride, and from about 3% to about 70%, by weight, of 100% formaldehyde, in (b) an aqueous medium, the concentration of said mixture in said solution being from about 1% to about 50%, wt/vol.
Claim 13
Dependent Claim
13 words
The composition of claim 9 wherein said aqueous medium is an aqueous alkanol.
Claim 14
Dependent Claim
10 words
The composition of claim 10 wherein the alkanol is methanol.
Claim 15
Dependent Claim
11 words
The composition of claim 9 wherein said aqueous medium is water.
Claim 16
Dependent Claim
11 words
The composition of claim 12 wherein said water contains a surfactant.
Claim 17
Dependent Claim
50 words
The composition of claim 9 wherein said mixture contains from about 75% to about 85%, by weight, of trichloroacetic acid, from about 2% to about 8%, by weight, of zinc chloride, and the balance formaldehyde, said mixture being in a 40 to 50%, wt/vol. solution of water containing a surfactant.
Claim 18
Dependent Claim
62 words
The composition of claim 14 wherein said mixture contains about 80%, by weight, of trichloroacetic acid, from about 5% to about 6%, by weight, of zinc chloride, and from about 14% to about 15%, by weight of 100% formaldehyde, said mixture being in a 45 to 50%, wt/vol., solution of water, said solution further containing up to 1% vol/vol., of a surfactant.
Claim 19
Dependent Claim
13 words
The composition of claim 15 wherein the surfactant is an alkylaryl polyether alcohol.
Claim 20
Dependent Claim
48 words
The composition of claim 9 wherein said mixture contains from about 30% to about 40%, by weight, of trichloroacetic acid, from about 10% to about 20%, by weight, of zinc chloride and the balance formaldehyde, said mixture being in a 1 to 5%, wt/vol. solution of aqueous methanol.
Claim 21
Dependent Claim
22 words
The composition of claim 17 wherein said solution also contains from about 1% to about 5%, vol./vol., of a hydrophillic polyethylene glycol.
Claim 22
Dependent Claim
19 words
The composition of claim 18 wherein the ratio of methanol to water is from about 2:1 to about 3:1.
Claim 23
Dependent Claim
54 words
The composition of claim 17 wherein said mixture contains about 32%, by weight, trichloroacetic acid, about 16%, by weight, of zinc chloride and about 52%, by weight, of formaldehyde, said mixture being in a 1.5 to 2.5%, wt/vol, solution of aqueous methanol wherein the ratio of methanol to water is from 2.4:1 to 2.5:1.
Claim 24
Dependent Claim
29 words
The composition of claim 20 wherein said solution also contains from about 2% to about 3%, vol/vol., of a hydrophillic polyethylene glycol having a molecular weight of about 200.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1979-12-14 | 1980-06-14 | 1980-12-14 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1983-12-14 | 1984-06-14 | 1984-12-14 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1987-12-14 | 1988-06-14 | 1988-12-14 | $7700.00 | Window Closed / Lapsed |