Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization

TECHNIQUES AND RESOURCES

Hybridization chain reaction enables a unified approach

to multiplexed, quantitative, high-resolution

immunohistochemistry and in situ hybridization

Maayan Schwarzkopf,

†

Mike C. Liu,

†

Samuel J. Schulte,

‡

Rachel Ives,

‡

N. Husain,

Harry M.T. Choi

2,*

and Niles A. Pierce

1,3,*

ABSTRACT

RNA in situ hybridization (RNA-ISH) based on the mechanism of

hybridization chain reaction (HCR) enables multiplexed, quantita-

tive, high-resolution RNA imaging in highly autofluorescent samples

including whole-mount vertebrate embryos, thick brain slices, and

formalin-fixed paraffin-embedded (FFPE) tissue sections. Here, we

extend the benefits of 1-step, multiplexed, quantitative, isother-

mal, enzyme-free HCR signal amplification to immunohistochem-

istry (IHC), enabling accurate and precise protein relative quanti-

tation with subcellular resolution in an anatomical context. More-

over, we provide a unified framework for simultaneous quantitative

protein and RNA imaging with 1-step HCR signal amplification

performed for all target proteins and RNAs simultaneously.

KEYWORDS: Immunofluorescence (IF), RNA fluorescence

in situ hybridization (RNA-FISH), qHCR imaging, formalin-

fixed paraffin-embedded (FFPE) mouse brain and human

breast tissue sections, whole-mount zebrafish embryos.

SUMMARY:

Signal amplification based on the mechanism of hy-

bridization chain reaction enables multiplexed, quantitative, high-

resolution imaging of protein and RNA targets in highly autofluo-

rescent tissues.

INTRODUCTION

Biological circuits encoded in the genome of each organism di-

rect development, maintain integrity in the face of attacks,

control responses to environmental stimuli, and sometimes

malfunction to cause disease.

RNA in situ hybridization

(RNA-ISH) methods (Gall & Pardue, 1969; Cox

et al.

, 1984;

Tautz & Pfeifle, 1989; Rosen & Beddington, 1993; Wallner

et al.

, 1993; Nieto

et al.

, 1996; Thisse & Thisse, 2008) and

immunohistochemistry (IHC) methods (Coons

et al.

, 1941;

Takakura

et al.

, 1997; Sillitoe & Hawkes, 2002; Ahnfelt-Ronne

et al.

, 2007; Psychoyos & Finnell, 2009; Ramos-Vara & Miller,

2014; Fujisawa

et al.

, 2015; Staudt

et al.

, 2015) provide biolo-

gists, drug developers, and pathologists with critical windows

into the spatial organization of this circuitry, enabling imag-

ing of RNA and protein expression in an anatomical context.

While it is desirable to perform multiplexed experiments in

which a panel of targets are imaged quantitatively at high

resolution in a single specimen, using traditional RNA-ISH

and IHC methods in highly autofluorescent samples including

whole-mount vertebrate embryos and FFPE tissue sections,

multiplexing is cumbersome, staining is non-quantitative, and

Division of Biology & Biological Engineering, California Institute of Technology,

Pasadena, CA 91125, USA.

Molecular Instruments, Inc., Los Angeles, CA 90041,

USA.

Division of Engineering & Applied Science, California Institute of Technology,

Pasadena, CA 91125, USA.

†

Authors contributed equally.

‡

Authors contributed

equally.

*Authors for correspondence (niles@caltech.edu and

harry@molecularinstruments.com)

spatial resolution is routinely compromised by diffusion of re-

porter molecules. These multi-decade technological shortcom-

ings are significant impediments to biological research as well

as to advancement of drug development and pathology assays,

impeding high-dimensional, quantitative, high-resolution anal-

yses of developmental and disease-related regulatory networks

in an anatomical context.

RNA-ISH methods detect RNA targets using nucleic acid

probes (Qian

et al.

, 2004; Silverman & Kool, 2007) and

IHC methods detect protein targets using antibody probes

(Ramos-Vara & Miller, 2014). In either case, probes can be

direct-labeled with reporter molecules (Kislauskis

et al.

, 1993;

Femino

et al.

, 1998; Levsky

et al.

, 2002; Kosman

et al.

, 2004;

Capodieci

et al.

, 2005; Chan

et al.

, 2005; Raj

et al.

, 2008),

but to increase the signal-to-background ratio, are more of-

ten used to mediate signal amplification in the vicinity of the

probe (Qian

et al.

, 2004; Silverman & Kool, 2007; Ramos-

Vara & Miller, 2014). A variety of in situ amplification ap-

proaches have been developed including immunological meth-

ods (Macechko

et al.

, 1997; Hughes & Krause, 1998; Kosman

et al.

, 2004), branched DNA methods (Collins

et al.

, 1997;

Bushnell

et al.

, 1999; Player

et al.

, 2001; Qian & Lloyd, 2003;

Wang

et al.

, 2012; Kishi

et al.

, 2019; Saka

et al.

, 2019), in situ

PCR methods (Wiedorn

et al.

, 1999; Qian & Lloyd, 2003),

and rolling circle amplification methods (Zhou

et al.

, 2001;

Schweitzer & Kingsmore, 2001; Larsson

et al.

, 2004; Zhou

et al.

, 2004; Larsson

et al.

, 2010). However, for both RNA-

ISH (Tautz & Pfeifle, 1989; Harland, 1991; Lehmann & Tautz,

1994; Kerstens

et al.

, 1995; Nieto

et al.

, 1996; Pernthaler

et al.

2002; Kosman

et al.

, 2004; Thisse

et al.

, 2004; Denkers

et al.

2004; Clay & Ramakrishnan, 2005; Barroso-Chinea

et al.

2007; Acloque

et al.

, 2008; Piette

et al.

, 2008; Thisse & Thisse,

2008; Weiszmann

et al.

, 2009; Wang

et al.

, 2012) and IHC

(Takakura

et al.

, 1997; Sillitoe & Hawkes, 2002; Ahnfelt-Ronne

et al.

, 2007; Psychoyos & Finnell, 2009; Ramos-Vara & Miller,

2014; Fujisawa

et al.

, 2015; Staudt

et al.

, 2015), traditional

in situ amplification based on enzyme-mediated catalytic re-

porter deposition (CARD) remains the dominant approach for

achieving high signal-to-background in highly autofluorescent

samples including whole-mount vertebrate embryos and FFPE

tissue sections. CARD is widely used despite three significant

drawbacks: multiplexing is cumbersome due to the lack of

orthogonal deposition chemistries, necessitating serial ampli-

fication for one target after another (Lehmann & Tautz, 1994;

Nieto

et al.

, 1996; Thisse

et al.

, 2004; Denkers

et al.

, 2004; Kos-

man

et al.

, 2004; Clay & Ramakrishnan, 2005; Barroso-Chinea

et al.

, 2007; T ́oth & Mezey, 2007; Acloque

et al.

, 2008; Piette

et al.

, 2008; Glass

et al.

, 2009; Stack

et al.

, 2014; Mitchell

et al.

, 2014; Tsujikawa

et al.

, 2017), staining is qualitative

rather than quantitative, and spatial resolution is routinely

The copyright holder for this preprint (which

this version posted June 2, 2021.

;

https://doi.org/10.1101/2021.06.02.446311

doi:

bioRxiv preprint