Fluorescent in situ hybridization technique for cell type identification and characterization in the central nervous system Akiya Watakabe*, a, b, Yusuke Komatsua, Sonoko Ohsawaa and Tetsuo Yamamoria, b a Division of Brain Biology, National Institute for Basic Biology, 38 Nishigonaka Myodaiji, Okazaki

444-8585, Japan b Department of Molecular Biomechanics, The Graduate University for Advanced Studies, 38

Nishigonaka Myodaiji, Okazaki 444-8585, Japan

This is an author preprint version of the article published in “Methods”, by Elsevier.

The article is available on line at

Fluorescent In Situ Hybridization technique for cell type identification and

characterization in the central nervous system

Akiya Watakabe, Yusuke Komatsu, Sonoko Ohsawa and Tetsuo Yamamori

Abstract

Central nervous system consists of a myriad of cell types. In particular, many subtypes

of neuronal cells, which are interconnected with each other, form the basis of functional

circuits. With the advent of genomic era, there have been systematic efforts to map

gene expression profiles by in situ hybridization (ISH) and enhancer-trapping strategy.

To make full use of such information, it is important to correlate “cell types” to gene

expression. Toward this end, we have developed highly sensitive method of fluorescent

dual-probe ISH, which is essential to distinguish two cell types expressing distinct

marker genes. Importantly, we were able to combine ISH with retrograde tracing and

antibody staining including BrdU staining that enables birthdating. These techniques

should prove useful in identifying and characterizing the cell types of the neural tissues.

In this article, we describe the methodology of these techniques, taking examples from

our analyses of the mammalian cerebral cortex.

1. Introduction

Central nervous system consists of a myriad of cell types, including neurons, glias,

endothelial cells, etc. On top of it, each cell type can be further subdivided into many

different subtypes [4,27]. Considering that the neuronal circuit is an assembly of

various neuronal types, the identification and characterization of each subtype is central

to the understanding of the circuit [13]. Recently, systematic efforts to map gene

expression in the brain, such as Allen Brain Atlas ([22]; http://www.brain-map.org/),

GENSAT ([11]; http://www.gensat.org/index.html) and others (e.g., genepaint.org;

http://www.genepaint.org/Frameset.html) have revealed many candidate marker genes

for cell type identification. Obviously, certain genes are specifically expressed by

particular subsets of neurons. But what are the common features of these neurons?

How are they related to the classical neuronal subtypes defined by morphology,

electrophysiological and pharmacological properties, antibody staining and connection

specificity? What exactly is “cell type” of neurons?

Our laboratory has been trying to identify the unique features of the primate

neocortex using molecular biological techniques. Specifically, we have been searching

for area- and/or layer-specific genes and using them as probes for comparative ISH

analyses [37,41]. What we considered critical in these analyses was the identification of

cell types, because, if we want to compare something across species, we need to

compare the same thing.

In the cerebral cortex, there are two fundamental cell types, excitatory and

inhibitory neurons [23]. These two types can be unambiguously identified by

expression of vesicular glutamate transporter 1 (VGluT1) and GABA or GABA

synthesizing enzyme GAD, respectively [10,33]. The subtypes of inhibitory neurons

can further be classified by expression of several well-known markers [4,7,17].

Because of such specific marker expression, antibody staining has been used

extensively to histologically identify these neuronal subtypes. However, some proteins

are not localized in the cell body (such as VGluT1) and difficult to be combined with

ISH. Furthermore, there are many potentially good marker genes, whose expressions

can be detected only by ISH due to lack of good antibodies. It is, therefore, desirable

that we can perform dual-probe ISH, in which we can directly compare the mRNA

expression of two genes simultaneously at cellular resolution.

Conceptually, dual-probe ISH is similar to immunofluorescent double staining

using two antibodies simultaneously. However, the former is often technically more

demanding, because the copy number of mRNA molecules could be very low and often

requires higher degree of amplification for visualization. The key for success depends

on the method of signal amplification. Initially, the detection in ISH was done by using

radioactive probes [25]. Then, non-radioactive method using haptens, such as biotin,

digoxigenin (DIG), and fluorescein (FITC) for probe labeling became more popular. In

a typical method, the hybridized DIG-labeled probe is detected by anti-DIG antibody

conjugated with alkaline-phosphatase, which catalytically converts the hybridization

signal to nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP)

precipitation. By using radioactive and non-radioactive probes for two genes, these

methods can be combined for double labeling. Another way for dual probe ISH is to

use different haptens to label two genes and detect them consecutively using the

substrates with different colors for alkaline phosphatase reaction (e.g., see [21]).

Although these and other methods of dual probe ISH have been used successfully for

some purposes, most of the methods lacked the resolution and sensitivity comparable to

the immunofluorescent double labeling. The only exceptions were those that used

tyramide signal amplification (TSA) technique (e.g., [19,20,40]).

TSA is one type of “CARD” or CAtalyzed Reporter Deposition technique

[35], in which the horse radish peroxidase (HRP)-conjugated anti-hapten antibody

catalyzes the deposition of another hapten, such as biotin, dinitrophenol (DNP), and

various fluorescent moieties to its near vicinity. Once the hybridization signal is

TSA-amplified, it can be converted to any fluorescent color (see Fig. 1). The

fluorescent detection of alkaline-phosphatase activity using HNPP/Fast Red as substrate

is also highly sensitive. Thus, at this point, we have several options to visualize the

hybridization signals fluorescently. With such advancement at hand, ISH can now be

combined with various other histological techniques.

In this paper, we describe the TSA-based dual probe ISH method, which is

useful to visualize diverse cell populations in the cerebral cortex and other brain regions.

We also describe the method to combine fluorescent ISH with retrograde tracing,

antibody staining and BrdU labeling. The identification of neuronal subtype is often

enigmatic because of diversity of neuronal phenotypes. It is also often the case that a

particular phonotype is not necessarily an all-or-none property and is a spectrum

between 0 and 1. Thus, to identify and characterize neuronal subtypes, it is essential to

define properties that are central to the “identity” of each neuron. The ISH-based

characterization, combined with various other techniques, has a promise to clarify the

complex issue of “cell type”. The protocols described here can be found in our past

studies [19,38] and are also available at our website

(http://www.nibb.ac.jp/brish/indexE.html).

2. Description of method

2.1 Overview

There are many variations of the ISH protocol. The implementation of the

fluorescent detection method described herein, however, should be applicable to most

protocols. In this paper, we first describe the methodology of free-floating dual probe

ISH, in which free floating sections are used for hybridization (chapter 2.2). We then

describe an alternative protocol, in which cryostat sections on a slideglass are used

(chapter 2.3). These two protocols use different procedures before the fluorescent

detection steps, and are suitable for different types of tissues. But the fluorescent

detection steps are essentially the same. In chapter 2.3, we also describe modification

of the basic two-color ISH to triple ISH. In chapter 2.4, we will describe how we can

combine the retrograde tracing with fluorescent ISH. We will explain the choice of the

tracer for ISH and show typical results obtained by this method. Depending on the

antibodies and antigens, it is also possible to combine ISH with immunohistochemsitry.

In chapter 2.5, we will explain some of the successful examples, among which we will

describe the method to combine BrdU birthdating with ISH. The reagents used in these

protocols are listed in Table 5.

2.2 Free-floating dual probe ISH with fluorescent detection

The original protocol for our free-floating ISH method came from the study

by Liang et al. [24]. In this protocol, the perfusion fixed brain tissue is sliced to 15-50 µm sections. The sections are postfixed with paraformaldehyde, digested with proteinase K for probe penetration, acetylated and hybridized with DIG-labeled

antisense RNA probe. After RNase treatment to remove non-specifically bound probes,

followed by several washes, the hybridized DIG-probe is detected by anti-DIG antibody

conjugated with alkaline-phosphatase, and visualized by NBT/BCIP color reaction. The

free-floating sections are mounted on a slideglass for observation, after all these

procedures. Generally speaking, free-floating method gives us better ISH images than

the ISH done on slideglasses. But the latter method also provides nice results and will

be described below.

To perform dual probe ISH, FITC-labeled antisense RNA probe is used

simultaneously with DIG-labeled probe in the hybridization solution. Instead of

NBT/BCIP color reaction, DIG and FITC probes are fluorescently detected in the

following manner (Fig. l). First, the FITC probe is detected by HRP-conjugated

anti-FITC antibody for TSA-DNP reaction followed by detection with anti-DNP

antibody conjugated with Alexa 488. Second, DIG probe is detected by

alkaline-phosphatase conjugated anti-DIG antibody, in the same manner as single ISH,

but we use HNPP/Fast Red as the substrate for red fluorescence. The step-by-step

protocol for this dual probe ISH method is presented in Table 1. Depending on the

abundance of the transcripts, and the cell-type specificity of mRNA expression, it can

rival the immunofluorescent double labeling (see Fig. 2 for an example of dual probe

ISH).

Key parameters for fluorescent detection The key point of this protocol is the

use of TSA. There are now many variations of TSA system, by which various haptens

and fluorophores can be deposited. In our hands, TSA-Plus (DNP) system worked most

consistently and sensitively for ISH. For abundant mRNAs, we had good results with

TSA-biotin (use streptavidin-Cy2 for detection). In both systems, the extent of signal

amplification is superb. However, because of the high degree of amplification, the

background noises (scattered granular speckles) tend to be amplified to obscure the true

hybridization signals. To avoid this, the quality of anti-FITC HRP antibody, as well as

its titration is most critical. The HRP antibody of some vendors did not work well even

after titration. 1/4000 dilution of the Jackson lab antibody (Table 5) generally gives

good results. But when granular backgrounds are high, the titration of the HRP

antibody needs to be empirically determined. The time for antibody binding also

requires caution. 30 min incubation time recommended in the manufacturer’s protocol

is too short to provide good signal-to-noise (S/N) ratio, even when the HRP antibody is

titrated. The incubation time for TSA reaction also affects the result. Generally

speaking, if the true hybridization signals are as strong as the background noises, the

S/N ratio would be dramatically improved by changing the above mentioned

parameters. When we perform dual probe ISH, there is a choice of which genes to label

with DIG and FITC. Also, although the protocol uses anti-FITC-HRP for TSA reaction

and anti-DIG alkaline phosphatase for HNPP/Fast Red, we can also use anti-DIG-HRP

for TSA and anti-FITC alkaline phosphatase for HNPP/Fast Red reaction. The choice

depends on the abundance of the genes to be tested. When the mRNA is abundant,

TSA-labeling provides an image with higher S/N contrast than HNPP/Fast Red.

However, due to granular nature of hybridization signals of TSA-staining, we feel that

the overall sensitivity of detection is better for HNPP/FastRed staining than

TSA-staining. As to the probe labeling, DIG label is, somehow, always better than

FITC label. So, depending on the combination of the genes, we need to switch

DIG/FITC label and TSA/HNPP Fast Red detection to get optimal staining.

Regarding the HNPP/Fast Red staining, it is useful to keep in mind that the

reaction products of HNPP/Fast Red easily get diffused. The mounting media is quite

critical. CC/Mount (Diagnostic Biosystems, India) that we are using is compatible with HNPP/Fast Red, but we had bad luck with other mounting media including

Fluoromount from the same manufacturer. Even when we use CC/Mount, we

occasionally find the HNPP/Fast Red signals to diffuse for some unknown reason. In

such cases, we repeat the reaction on the slideglass. Once we get good preparation, the

red stain is stable for months, when stored frozen. Nevertheless, we recommend taking

photos as soon as possible. Compared with TSA-staining, HNPP/Fast red staining

exhibits uniform but high tissue background and requires thin section (15-20 µm) for

good images. Confocal imaging will be required to process thicker sections.

Key parameters of in situ hybridization in general The success of the

dual-probe ISH, of course, depends on the quality of the ISH itself. Here we explain

some of the important parameters for ISH in general. First, the quality of the tissue

sections for ISH is important. Good perfusion fix with 4 % paraformaldehyde is

required for optimal signals. Uneven perfusion due to clumsy manipulation during

cardiac infusion can potentially result in uneven staining. The fixation solution could

also contain picric acid and/or glutaraldehyde. We had good result with a brain fixed in

4% paraformaldehyde, 0.2% picric acid, 0.1% glutaraldehyde in 0.1M PB. The

over-fixation, however, decreases the signals.

Regarding the ISH conditions, there are two important parameters that need to

be determined empirically. First, the concentration of the proteinase K greatly affects

the strength of the ISH signals. Generally speaking, the higher, the concentration, the

stronger, the signals. However, because the sections become very fragile after

proteinase K treatment, we must determine the optimal condition that gives strong

enough signals, but retains the integrity of the tissue sections. We routinely use 0.5

µg/ml proteinase K for adult mouse brain and 5 µg/ml for adult monkey brain, but this

must be empirically determined. Second, the hybridization temperature needs to be

adjusted for each probe. Practically speaking, most probes work nicely with 60 ℃

hybridization. If the probe is GC-rich and/or contain some unusual sequences, high

background noise may appear. In such cases, raising the hybridization temperature to

65, 68 and 72 ℃ would help.

About the probe We make antisense RNA probes by standard in vitro transcription

using T3/T7/SP6 RNA polymerase. The standard length of the cDNA used for probe

production is 500-1000 base pair. Although some other protocols suggest short RNA

probes (~100 nt) for ISH, the penetration into the tissue section seems to be efficient up

to 1000 nt in our protocol. However, long probes (>2000 nt) need to be degraded by

alkaline hydrolysis for efficient hybridization in the floating in situ method (for ISH on

slideglass, it is not necessary). Because the sensitivity of ISH increases with the length

of the probe, we routinely make multiple probes covering different regions of the gene

and use them mixed. When using multiple probes, it is necessary to confirm that each

probe exhibits the same hybridization profile. Actually, this is a good method to

confirm the specificity of hybridization.

The specificity of hybridization is always a matter of concern in the ISH

experiments. Non-specific hybridization could occur in the GC-rich region. There

could also be cross-hybridization to similar sequences. Perform BLAST

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) search at NCBI to check for cross hybridization

with the family genes. Based on ISH experiments using mouse and monkey specific

probes for the same gene, we think that sequence identity of 80 % is a reasonable line to

judge the possibility of cross hybridization. It must be kept in mind, however, that

cross-hybridization is not completely predictable. Control experiments by sense probe,

comparison of multiple probes, and changing hybridization temperature are all useful to

judge the credibility of the hybridization specificity.

2.3 Alternative protocol

In this section, we cover two topics. First, we describe an alternative protocol for ISH,

which is performed on a slideglass. Second, we describe TSA-biotin system, which can

serve as an alternative to TSA-DNP system or can be used to add one more color to

achieve triple ISH.

ISH on a slideglass Although free-floating ISH provides excellent images for

relatively “hard” adult brains, it is not suitable for “soft” brains of embryos and

neonates. It is also not suitable to make serial sections of olfactory bulb, spinal cord

and cerebellum, because of uniformity and small size. When the tissue sections are

prepared on a slideglass by a cryostat, we use the protocol by Schaeren-Wiemers et al.

[34] with slight modifications. Typically, the fresh dissected brain tissue is quick-frozen in Tissue-Tek OCT compound 4583 (Sakura Finetechnical Co. Ltd. Tokyo,

Japan) using liquid nitrogen cooled 2-methylbutane isopentan and 10-20 µm sections

are collected on a slideglass using cryostat. The tissue sections on a slideglass are

postfixed in 4% paraformaldehyde/PBS, acetylated and hybridized with the antisense RNA probe at 72 ℃ overnight. After hybridization, the slideglasse is washed three

times in 0.2xSSC at 72 ℃, blocked in 1 % blocking buffer and processed for DIG/FITC

detection as in the floating method. The step-by-step protocol for this slideglass method

is presented in Table 2.

A characteristic feature of this method is that high S/N ratio and probe penetration is achieved by hybridization at high temperature (72 ℃). The procedure is

simple and the sensitivity is good. Unless the tissue is “hard”, neither proteinase K nor

RNAse treatment is required. Despite such simplicity, slideglass method provides

excellent images of fluorescent dual probe ISH, although the probe does not penetrate

as deep as the floating method and may be less sensitive.

Using TSA-biotin system Although we had best results with TSA-Plus (DNP)

system, the kit is very expensive. A more economical alternative is TSA-biotin system,

in which biotin is deposited to the tissues. It can be used in place of TSA-Plus (DNP)

and fluorescent detection can be done by incubation with streptavidin-Cy2 or other

fluorescent reagents. TSA-biotin system can be easily made in the lab (see, for example

[14]). Although TSA-biotin system is less sensitive and finicky compared with TSA-DNP system, we had good results for abundant transcript using the in-house

reagents.

Using different kinds of TSA-system, it is possible to perform triple-probe

ISH [30,38]. Briefly, three genes are labeled with DIG, FITC and biotin and used for simultaneous hybridization. First, the FITC signal is converted to DNP by TSA-DNP as

in dual-probe ISH. Subsequently, the bound antibody is removed by 0.1 M glycine-HCl

(pH 2.2), 0.1 % Tween 20 as in [21]. The sections are blocked again in 0.5 % TNB, which is the blocking reagent that comes together with the TSA-DNP kit. The biotin-labeled probe is, then, detected by anti-biotin antibody, conjugated with HRP, and processed for TSA-biotin reaction. In the final visualization step,

anti-DIG-alkaline phosphatase, anti-DNP-Alexa488 and streptavidin-Alexa350 are

incubated with the sections and processed for HNPP/Fast Red reaction. For some

unknown reason, the biotin-label detection sometimes fails. But if the protocol works,

the co-expression of three genes can be determined.

2.4 Tracer ISH

Invention of anterograde and retrograde tracers for tract-tracing greatly contributed to

the elucidation of neural connectivity [18]. The retrograde tracer, in particular, provides an important link between cell type and connections. For example, if you inject a

retrograde tracer into thalamus, a subset of neurons in layer 6 of the cerebral cortex are

lit up (Fig. 3). These thalamic projecting neurons take up the tracer at their nerve

terminals in the thalamus, and the tracer is transported back to the cell body. In the

cerebral cortex, the projection specificity is a key property of a distinct class of neurons.

For example, layer 5 neurons projecting to subcortical nuclei and contralateral cortex

have been long recognized to exhibit differential morphology and electrophysiological

properties [15,26]. Similarly, layer 6 neurons projecting to thalamus and claustrum

have differential morphology and potentially distinct physiological properties [6,16,43].

These studies suggest that the projection specificity may be a key “identifier” of cortical

cell type. Indeed, there is now evidence to believe that expression of certain genes is

correlated with projection specificity [1,2,12,38,42]. The flourish of molecular

information is now available in the literature and in public database. Of particular note

are the large-scale ISH database of Allen Brain Institute (http://www.brain-map.org/),

and BAC-transgenic database of GENSAT (http://www.gensat.org/index.html). Due to

these systematic efforts and other molecular biological works (e.g., [27,29]), there are

many candidate marker genes for cell-type identity. It is thus quite important that we

can combine retrograde tracing with ISH.

The success of tracer-ISH experiment depends on whether the tracer can

withstand the rather harsh treatment of ISH or not. In our previous work, we have

shown that FastBlue can be used for tracer-ISH experiment [38]. Because FastBlue shows blue fluorescence, it can be combined with single or double ISH without any

changes in the protocol. However, the fluorescence of FastBlue diminishes

considerably during ISH. Other choice of tracer includes FluoroGold and cholera toxin

B fragment (CTB) conjugated with Alexa dyes. Not only can they withstand ISH

treatment, their signals can be enhanced by antibody against FluoroGold or Alexa dyes

after ISH. In Table 3, we show a model protocol for FluoroGold-ISH. In this protocol,

the FluoroGold signal is enhanced by antibody detection and ISH signal is visualized by

HNPP/Fast Red. The ISH detection step can be modified to provide green color by

using TSA, if desired. In Fig. 3, we show an example of such tracer-ISH experiment.

In this example, lack of Nurr1 mRNA in the corticothalamic layer 6 neurons is clearly

demonstrated, consistent with the previous tracer-immunofluorescence study [2]. We

believe that this kind of analyses is important for integrative understandings of the “cell

type” in the cerebral cortex.

2.5 Combination with antibody staining (BrdU birthdating)

There are many good antibodies that provide useful information about the anatomical

structure of the neural tissues. Despite rather harsh tissue treatment of the ISH

procedure, it is often possible to combine antibody staining with ISH. Although many

good monoclonal antibodies (such as SMI32) were not suitable for

immunofluorescence-ISH, we had success with some polyclonal antibodies such as

those against paravalbumin and calbindin, among others. The success seems to depend

on the nature of the antibody and there is not much we can control to enable

immunostaining of the ISH processed samples. One of the few parameters that can be

changed is the selection of the blocking reagent. 1 % Blocking Reagent (Roche

Diagnostics) used for ISH is often too strong: skim milk or other reagents used for

normal immunohistochemistry should provide better results for immunostaining. If the

antibody of interest is not compatible with ISH, one possibility is to produce a

compatible antibody de novo. Interestingly, Nakamura et al. succeeded in producing the

anti-GFP antibody suitable for double staining with ISH by modifying the antigen

preparation step for immunization [28]. Their method is worth considering if

combining immunostaining with ISH is required.

As a successful example of combining immunostaining with ISH, here we

provide a protocol to combine BrdU birthdating with ISH. Because the specification of

cortical neurons occurs according to the developmental timetable, labeling neurons in

their final cell division at a particular embryonic period has been a useful technique to

investigate corticogenesis. Originally, it was done by injecting 3H-thimidine to the

pregnant animal and observing the incorporation of the radioactivity in the nuclei of the

cortical neurons that were born at the time of injections [5,31]. Recently, injection of

thymidine analog, BrdU (bromodeoxyuridine), followed by anti-BrdU immunostaining

[9] has become a popular alternative. The same technique is used to examine the

neurogenesis in the adult brain [3,9].

To combine BrdU birthdating with ISH, one obstacle was the denaturation

step before anti-BrdU immunostaining, which is required to expose the BrdU

incorporated into the chromosomal DNA. After this step, the hybridized probe and/or

bound antibodies are released and lost from the site. To overcome this obstacle, we

used the TSA system to convert the DIG hybridization signal to DNP, before the BrdU

immunodetection step (Table 4). By this protocol, we succeeded in combining BrdU

labeling with ISH (Fig. 4). It is also possible to perform the double staining by first

colorizing the ISH signal to NBT/BCIP as in normal ISH and then perform BrdU

staining. Either way, the correlation of birthdate and gene expression profile is an

important information to consider “cell type” of the cerebral cortex.

3. Concluding remarks

In the field of developmental neuroscience, it has been considered that the fate of

cortical neurons is specified early during development to later acquire characteristic

features of each “cell type” (e.g., [8,32,36,39]). Although recent studies are rapidly

unraveling the molecular genetic mechanism of cortical cell type specification (for a

review, [27]), the significance of each cell type within the cortical circuit still remains

elusive. In a way, we still do not know what “cell type” exactly means [4,26,27,29,37].

By using the fluorescent ISH techniques that we described in this article, we will better

understand the physiological meaning of gene expression, which will, in turn, help to

elucidate normal neurological function.

Acknowledgements

We thank Drs. Fengyi Liang and Tsutomu Hashikawa for teaching us their floating in

situ methods when we started our study. We also thank Dr. Takashi Kitsukawa for

instructing the slideglass method. We thank helpful information of Drs. Hiroyuki Hioki

and Takeshi Kaneko as well as Drs. Noritaka Ichinohe and Kathleen Rockland.

Supported by the grant from the JSPS (KAKENHI19500304 and 22500300).

Table 1: protocol for free-floating dual probe ISH <DAY1> Cut the section and postfix in 4% paraformaldehyde/0.1MPB 4℃ overnight <DAY2> 0.1M PB 10 min x 2 0.75% Glycine/0.1M PB 15 min x 2 0.3% Triton X100/0.1M PB 20 min 0.1M PB 5 min Proteinase K (0.5-5 µg/ml) in PK buffer 37℃ 30 min Acetylation Buffer 10 min 0.1M PB 10 min x 2 Hybridization sol. 60℃ 1 hour Hybridization sol.+RNA probes (0.5~1 µg/ml total) Denature the probe at 80 ℃ for 5 min before adding to Hybridization sol.

60℃ overnight

<DAY3> 2xSSC/50% formamide/0.1% N-lauroilsarcosine (NLS) 60℃ 15~20 min x 2 RNase buffer 5 min RNase A (20 µg/ml) treatment in RNase buffer 37℃ 30 min 2xSSC /0.1% NLS 37℃ 15~20 min x 2 0.2xSSC/0.1% NLS 37℃ 15~20 min x 2 TS7.5 5 min

***fluorescent detection*** 1% Blocking reagent/TS7.5 30 min~1 hour 1/4000 anti-FITC-HRP in 1% Blocking reagent/TS7.5 2~5 hours or 4℃ overnight TNT 15 min x 3 TSA-Plus (DNP) 30 min TNT 10 min x 3 1/1000 anti-DIG-AP, 1/500 anti-DNP Alexa488 in 1% Blocking reagent/TS7.5

2~5 hours or 4℃ overnight

<DAY4> TNT 15 min x 3 TS8.0 10 min HNPP/Fast Red in TS8.0 20~30min PBS-EDTA 3~5 minx2 Hoechst 33342 (1µg/ml) in PBS-EDTA 5 min PBS-EDTA 3~5 min x 2 Mount on slideglass and envelop in CC/Mount

Table 2: protocol for dual probe ISH on a slideglass (Schaeren-Wiemers et al. 1993 [34]) <DAY1>

Cut the section and save on a slideglass Can be stored at –80 ℃ 4% paraformaldehyde 10 min

PBS 3 min x 3

(Proteinase K (0.5~5 µg/ml) in PK buffer)# 37 ℃ 30 min For fixed tissue

(4% paraformaldehyde)# 5 min For fixed tissue

(PBS)# 3 min x 3 For fixed tissue

Acetylation buffer 10 min

(0.3% Triton X100/0.1M PB)# 20 min For fixed tissue

PBS 5 min x 3

SW Hybridization sol. Room temp briefly

SW Hybridization sol.+RNA probe (0.5~1 µg/ml total) 72 ℃ overnight

<DAY2>

0.2 x SSC 72 ℃ 30 min x 3

TS7.5 5 min

***fluorescent detection***

1% Blocking reagent/TS7.5 1hour

1/4000 anti-FITC-HRP in 1% Blocking reagent/TS7.5 2~5 hours or 4℃ overnight

TNT 10 min x 3

TSA-Plus (DNP) $ 3~10 min

TNT 5 min x 3

1/1000 anti-DIG-AP, 1/500 anti-DNP-Alexa488 in 1% Blocking reagent/TS7.5

2~5 hours or 4℃ overnight

<DAY4>

TNT 10 min x 3

TS8.0 5 min

HNPP/Fast Red in TS8.0 20~40min

PBS-EDTA 3~5 minx2

Hoechst 33342 (1µg/ml) in PBS-EDTA 5 min

PBS-EDTA 3~5 min x 2

Envelop in CC/Mount Don’t dry

# skip these procedures for non-fixed tissues.

$ Dilute the already diluted TSA reagents two fold with distilled water.

Table 3: protocol for Tracer-ISH (Fluoro Gold) <DAY1~3> Same as normal ISH before blocking The following is the protocol for DIG-labeled probe

***fluorescent detection*** 2% skim milk/TS7.5 30 min 1/500 anti-FluoroGold in 2% skim milk/TS7.5

2~5 hours or 4℃ overnight

TNT 15 min x 3 1/1000 anti-DIG-AP, 1/500 anti-rabbit-Cy2 in 1% Blocking reagent/TS7.5

2~5 hours or 4℃ overnight

<DAY4> TNT 15 min x 3 TS8.0 10 min HNPP/Fast Red in TS8.0 20~40min PBS-EDTA 3~5 minx2 Hoechst 33342 (1µg/ml) in PBS-EDTA 5 min PBS-EDTA 3~5 min x 2 Mount on slideglass and envelop in CC/Mount

Table 4: protocol for BrdU-ISH

<DAY1~3> Injection of BrdU solution into pregnant animals (50 mg/kg, i.p.) Same as normal ISH before blocking The following is the protocol for DIG-labeled probe

***fluorescent detection*** 1% Blocking reagent/TS7.5 30 min-1hour 1/2000 anti-DIG-HRP in 1% Blocking reagent/TS7.5

2~5 hours or 4℃ overnight

TNT 15 min x 3 TSA-Plus (DNP) 30 min TNT 10 min x 3 1.5N HCl (for denaturation) 37℃ 30 min TNT 5 min x2 5% skim milk/PBST 20 min 1/75 anti-BrdU in 5% skim milk/PBST 4℃ overnight <DAY4> PBS 10min x 3 1/500 anti-rat-biotin in 5% skim milk/PBST 2~5 hours or 4℃ overnight

TNT 10 min x 3 ABC kit (sol A:1/100, sol B:1/100, in TNT) 30 min TNT 10 min x3 1/500 Streptavidin-Cy3, 1/500 anti-DNP-Alexa488 in 5% skim milk/PBST

2~5 hours or 4℃ overnight

TNT 10 min x 3 Mount on slideglass and envelop in CC/Mount

Table 5: Reagents list <Buffers> PK buffer 0.1 M Tris.HCl (pH8.0), 50 mM EDTA

Acetylation Buffer

0.1 M triethanolamine 169.7 ml +0.3 ml HCl, Add 10 µl of Acetic anhydride per 4 ml just before use

Hybridization sol.

Mix the following solutions: 20xSSC; 12.5 ml, 10 % Blocking reagent; 10 ml, Formamide; 25 ml, 2% N-lauroylsarcosine; 2.5 ml, 10 % SDS; 0.5 ml

SW Hybridization sol.

5xSSC, 50 % Formamide, 5xDenhardt’s, 250 µg/ml yeast tRNA, 500 µg/ml salmon sperm DNA

RNase buffer 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 M NaCl

TS7.5 0.1 M Tris-HCl, pH7.5, 0.15 M NaCl TS8.0 0.1 M Tris-HCl. PH 8.0, 0.1 M NaCl , 10 mM MgCl2

TNT TS7.5, 0.05% Tween20

PBS-EDTA PBS with 10 mM EDTA

BrdU solution (20 mg/ml) BrdU (sigma#B5002) in 0.007 N NaOH, 0.9% NaCl PBST PBS with 0.2 % TritonX100 <Reagents> Proteinase K Roche Diagnostics: Proteinase K #3115887

Blocking Reagent Roche Diagnostics: Blocking Reagent #11 096 176 001

anti-FITC-HRP

Jackson ImmunoResearch laboratory: Peroxidase-IgG Fraction Monoclonal Mouse Anti-FITC #200-032-037

TSA-Plus (DNP) Perkin Elmer: #NEL747A: Make working solution as instructed

HNPP/Fast Red Roche Diagnostics: #1 758 888: Make working solution as instructed anti-DIG-AP

Roche Diagnostics: Anti-Digoxigenin-AP Fab fragments 11 093 274 910

anti-DNP Alexa488 Molecular Probe: #A-11097 Hoechst 33342 Dojindo: 346-07951

CC/Mount Diagnostic Biosystems: #K002

FluoroGold Biotium, Inc: #80014

Anti-FluoroGold Chemicon: AB153 rabbit anti-Fluorogold Polyclonal antibody Anti-BrdU Abcam: BrdU antibody [BU1/75 (ICR1)]

Elite ABC kit Vectastain: PK-6100

Streptavidin-Cy3 Jackson ImmunoResearch laboratory: #016-160-084

Fig.1 The scheme for Fluorescent double ISH

(Top panel) Digoxigenin (DIG) and FITC (fluorescein)-labeled antisense RNA is

hybridized simultaneously to the target mRNAs. (Middle panel) FITC is recognized by

anti-FITC antibody conjugated to HRP (horse radish peroxidase), which catalyze TSA

reaction, in which the free radical form of DNP-Tyramide reacts to be deposited to the

nearby tissue. (Bottom panel) DIG signal is converted to red fluorescence by alkaline

phosphatase activity of the anti-DIG-AP antibody. FITC signal is converted to green

fluorescence by anti-DNP antibody conjugated to Alexa 488.

Fig. 2 Dual probe fluorescent ISH of adult mouse cortex

DIG-labeled GAD67 and FITC-labeled VGluT1 probes were used to identify inhibitory

and excitatory neurons, respectively, in the adult mouse cortex. DIG probe was

visualized by alkaline phophatase reaction using HNPP/Fast Red as substrate and FITC

probe was visualized by anti-DNP Alexa 488 followed by TSA-Plus (DNP) reaction.

Note complete segregation of these signals. Bar: 100µm.

Fig. 3 Tracer-ISH experiment to demonstrate projection specificity of Nurr1-positive

neurons in the mouse cortex

FluoroGold was injected into thalamus, taken up at the nerve teminals by the thalamic

projecting neurons and was then transported back to the cell body. In this photo, the

thalamic projecting neurons in the cortex (corticothalamic neurons) were revealed after

ISH by anti-FluoroGold antibody (green). Nurr1 mRNA was visualized by fluorescent

ISH (red). Consistent with the antibody study [2], Nurr1 mRNA was not expressed in

corticothalamic neurons in layer 6. Bar: 100µm.

Fig. 4 BrdU bithdating combined with ISH

BrdU solution was injected into pregnant rat at E15 and the brain was perfusion-fixed at

P15. ISH of cholecystokinin (CCK) gene (green) was performed in combination with

BrdU immunostaining (red) as in Table 4. The white arrow indicates the double

positive cell for both CCK mRNA and BrdU immunostaining. Note that BrdU staining

reveals the nuclei of the labeled cells, whereas the mRNAs are localized in the cell body.

Bar: 100 µm.

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