# US 6,409,774 B1 · Electrophoresis-assisted staining of materials

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- Number: US 6,409,774 B1
- Document ID: US6409774B1
- Status: Granted
- Kind: B1
- Application number: 09/330,400
- Filed: 1999-06-11
- Granted: 2002-06-25
- Earliest claimed: 1999-06-11
- Assignees: Resolution Sciences Corporation
- Inventors: Russell L. Kerschmann; Ronald Odom; Thomas Kuwahara; Mark Reddington
- Web page: https://patentgrep.com/patent/US6409774B1

## CPC

- G01N 33/561: Immunoelectrophoresis
- G01N 1/31: Apparatus therefor
- G01N 27/44704: {Details; Accessories}
- G01N 27/44726: {using specific dyes, markers or binding molecules}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

A method for en bloc staining a biological sample by (a) immersing the sample in a staining solution including an ionically conductive solution and a charged stain molecule or stain precursor that associates with a component of the sample; and (b) applying an electric field across the staining solution, whereby the stain molecule penetrates into the sample. Staining times are reduced.

## Claims

### Claim 1

A method for en bloc staining a sample, said method comprising the steps of:
a) immersing the sample in a staining solution including (i) an ionically conductive solution, and (ii) a charged stain molecule and/or stain precursor that associates with a component of the sample;
b) applying an electric field across the staining solution, whereby the stain molecule penetrates into the sample to produce an en bloc stained sample; and
c) embedding said en bloc stained sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the stain precursor is selected from the group consisting of antibodies, nucleic acid probes and lectins.

### Claim 3 (depends on 2)

The method of claim 2, wherein the stain precursor is a primary antibody.

### Claim 4 (depends on 3)

The method of claim 3, further comprising the steps of:
c) immersing the primary antibody-containing sample in a staining solution including an tonically conductive solution and a charged secondary antibody that binds to the primary antibody; and
d) applying an electric field across the staining solution, whereby the secondary antibody penetrates into the sample.

### Claim 5 (depends on 3)

The method of claim 3, further comprising the step of:
immersing the primary antibody-containing sample in a solution comprising a secondary antibody that binds to the primary antibody.

### Claim 6 (depends on 4, 5)

The method of claim 4 or 5, wherein the secondary antibody is labeled, said label comprising a component for producing coloration in the sample.

### Claim 7 (depends on 6)

The method of claim 6, wherein the label comprises a fluorescent probe.

### Claim 8 (depends on 6)

The method of claim 6, wherein the stain molecule comprises an enzyme reactive to form a permanent colored product.

### Claim 9 (depends on 1)

The method of claim 1, wherein the stain molecule comprises a labeled carrier molecule, said carrier molecule selected from the group consisting of antibodies, nucleic acid probes and lectins and said label comprising a component for producing coloration in the sample.

### Claim 10 (depends on 9)

The method of claim 9, wherein the label comprises a fluorescent probe.

### Claim 11 (depends on 9)

The method of claim 9, wherein the stain molecule comprises an enzyme reactive to form a permanent colored product.

### Claim 12 (depends on 1)

The method of claim 1, further comprising the steps of:
infiltrating the sample with a gel support prior to immersing the sample in the staining solution.

### Claim 13 (depends on 12)

The method of claim 12, wherein the gel support is selected from the group consisting of agarose and polyacrylamide.

### Claim 14 (depends on 1)

The method of claim 1, wherein the staining solution is contained in a chamber and the sample substantially occupies and conforms to the cross-sectional area of the chamber.

### Claim 15 (depends on 1)

The method of claim 1, wherein a voltage in the range of 100-400V is applied.

### Claim 16 (depends on 15)

The method of claim 15, wherein a voltage in the range of 50-700V is applied.

### Claim 17 (depends on 1)

The method of claim 1, wherein the electric field is maintained for a time in the range of 1 to 25 hours.

### Claim 18 (depends on 1)

The method of claim 1, wherein the electric field is maintained in the range of 3 to 15 hours.

### Claim 19 (depends on 1)

The method of claim 1, further comprising the step of (d) sectioning said embedded en bloc stained sample.

### Claim 20

A method for reducing unbound stain molecule in a sectioned tissue, said method comprising the steps of:
a) immersing the a tissue sample in a staining solution including an ionically conductive solution and a stain precursor that associates with a component of the sample; and
b) applying an electric field across the staining solution, whereby penetration of the stain precursor into the sample and emigration of unbound stain precursor from the sample occurs.