Download Fluorescent in situ hybridization technique for cell type identification

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.
Hybridization sol.+RNA probes (0.5~1 µg/ml total)
Denature the probe at 80 ℃ for 5 min before adding to
Hybridization sol.
60℃ 1 hour
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
(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
PBS
5 min x 3
SW Hybridization sol.
Room temp briefly
SW Hybridization sol.+RNA probe (0.5~1 µg/ml total)
72 ℃ overnight
For fixed tissue
For fixed tissue
<DAY2>
0.2 x SSC
72 ℃ 30 min x 3
TS7.5
5 min
***fluorescent detection***
1% Blocking reagent/TS7.5
1/4000 anti-FITC-HRP in 1% Blocking reagent/TS7.5
1hour
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
Hoechst 33342 (1µg/ml) in PBS-EDTA
3~5 minx2
PBS-EDTA
3~5 min x 2
5 min
Envelop in CC/Mount
# skip these procedures for non-fixed tissues.
$ Dilute the already diluted TSA reagents two fold with distilled water.
Don’t dry
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
1/500 anti-FluoroGold in 2% skim milk/TS7.5
30 min
2~5 hours or 4℃ overnight
TNT
1/1000 anti-DIG-AP, 1/500 anti-rabbit-Cy2 in 1%
Blocking reagent/TS7.5
15 min x 3
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
1/2000 anti-DIG-HRP in 1% Blocking reagent/TS7.5
30 min-1hour
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
1/500 anti-rat-biotin in 5% skim milk/PBST
10min x 3
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
2~5 hours or 4℃ overnight
1/500 Streptavidin-Cy3, 1/500 anti-DNP-Alexa488
in 5% skim milk/PBST
TNT
Mount on slideglass and envelop in CC/Mount
10 min x 3
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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