# US 11,299,770 B2 · On-slide staining by primer extension

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,299,770 B2
- Document ID: US11299770B2
- Status: Granted
- Kind: B2
- Application number: 16/240,463
- Filed: 2019-01-04
- Published: 2019-10-31
- Granted: 2022-04-12
- Earliest claimed: 2014-06-23
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Nikolay Samusik; Garry P. Nolan; Yury Goltsev
- Web page: https://patentgrep.com/patent/US11299770B2

## CPC

- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method for analyzing planar sample is provided. In some cases the method comprises: (a) incubating the planar sample with a capture agent that is linked to an oligonucleotide, wherein the capture agent specifically binds to complementary sites in the planar sample; (b) reading a fluorescent signal caused by extension of a primer that is hybridized to the oligonucleotide, using fluorescence microscopy. Several implementations of the method, and multiplexed versions of the same, are also provided.

## Claims

### Claim 1

A composition comprising:
(a) a biological sample; and
(b) a plurality of antibodies;
wherein:
i. the plurality of antibodies are linked to single-stranded oligonucleotides, and
ii. the plurality of antibodies are chemically cross-linked directly to the biological sample.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the plurality of antibodies are crosslinked to the biological sample via a bifunctional cross-linker.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the plurality of antibodies are crosslinked to the biological sample via an amine-to-amine crosslinker.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the plurality of antibodies are crosslinked to the biological sample by formaldehyde.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the plurality of antibodies are crosslinked to the biological sample by a disuccinimdyl crosslinker.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the biological sample is planar.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the biological sample is a tissue section.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the biological sample is a formalin-fixed paraffin embedded (FFPE) tissue section.

### Claim 9 (depends on 1)

The composition of claim 1, wherein the biological sample is a tissue biopsy.

### Claim 10 (depends on 1)

The composition of claim 1, wherein the plurality of antibodies are bound to diagnostic markers in the biological sample.

### Claim 11 (depends on 1)

The composition of claim 1, wherein the plurality of antibodies comprises a first antibody linked to a first single-stranded oligonucleotide and a second antibody linked to a second single-stranded oligonucleotide.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the plurality of antibodies comprises at least 10 antibodies, each linked to a different oligonucleotide.

### Claim 13 (depends on 1)

The composition of claim 1, wherein the plurality of antibodies comprises at least 50 antibodies, each linked to a different oligonucleotide.

### Claim 14 (depends on 1)

The composition of claim 1, wherein a single-stranded oligonucleotide of the single stranded oligonucleotides is linked to a second oligonucleotide strand to form a double-stranded oligonucleotide.

### Claim 15 (depends on 14)

The composition of claim 14, wherein the double-stranded oligonucleotide comprises an overhang.

### Claim 16 (depends on 1)

The composition of claim 1, wherein one or more antibodies of the plurality of antibodies are fluorescently labeled.

### Claim 17 (depends on 16)

The composition of claim 16, wherein the fluorescent label is attached to an oligonucleotide that is linked to an antibody.

### Claim 18 (depends on 1)

The composition of claim 1, wherein the plurality of antibodies are monoclonal antibodies.

### Claim 19 (depends on 1)

The composition of claim 1, wherein the single stranded oligonucleotides linked to the plurality of antibodies are at least 10 nucleotides in length.

### Claim 20 (depends on 1)

The composition of claim 1, wherein the single stranded oligonucleotides linked to the plurality of antibodies are in the range of 15-200 nucleotides in length.