PerkinElmer, Inc.

™

TSA PLUS Biotin KIT
Tyramide Signal Amplification
For in Situ Hybridization and Immunohistochemistry

NEL749A001KT TSA Plus Biotin
Kit (50 - 150 slides*)NEL749B001KT TSA Plus Biotin
Kit (250 - 750 slides*)* number of slides determined byvolume used per section1For Laboratory Use
CAUTION: A research chemical
for research purposes only.

2TABLE OF CONTENTSI.

INTRODUCTION
A.

B.
C.

II.

PROTOCOL FOR ISH
A.
B.

C.

III.

Overview Protocol for TSA Plus Biotin for
In Situ Hybridization 10
Suggested ISH Protocol 11 First Time
Users 11 Controls 12 Reagent Titration
12 Quenching Endogenous Peroxidase 13
Volumes 14 Technical Support 14
Standard ISH Protocol 15 Preparation of
Buffers and Reagents 16 Procedural Notes
16 Step by Step Protocol 17

PROTOCOL FOR IHC
A.
B.

C.

IV.

Background Information 5 What is ISH? 5
What is IHC? 5 What is TSA? 5 How does
TSA™ Plus Biotin Signal Amplification
work? 6 What ISH and IHC mediums arecompatible with TSA Plus biotin? 6
The TSA Plus Biotin Kit 7 Intended Use 7
Safety Note 7
Components of TSA Plus Biotin Kits 8
Storage and Stability 8 Critical Reagents
Required But Not Supplied 9

Overview Protocol for TSA Plus Biotin
Immunohistochemistry 20
Suggested IHC Protocol 21 First Time
Users 22 Controls 22 Reagent Titration
22 Quenching Endogenous Peroxidase 23
Volumes 24 Technical Support 24
Standard IHC Protocol 24 Preparation of
Buffers and Reagents 24 Procedural Notes
26 Step by Step Protocol 27

TROUBLESHOOTING GUIDE
A.
B.
C.

In Situ Hybridization (ISH) 31
Immunohistochemistry (IHC) 32
Customer Technical Support Services 33

3

V.

ORDERING INFORMATION

35

VI.

REFERENCES

40

4

I. INTRODUCTION
A. Background Information
What is ISH?
In situ hybridization (ISH) is a technique used to detect,visualize and localize DNA and RNA at the cellular level.
Labeled probes are hybridized to nucleic acid targets in tissue
or cell preps. Probes can be labeled using common labelingtechniques such as in vitro transcription, nick translation, and3' end labeling. Detection schemes for in situ hybridization
include autoradiography for radioactive probes, dye deposition
for enzyme or hapten-labeled probes, and fluorescence forfluorophore-labeled probes.
What is IHC?
Immunohistochemistry (IHC) is a technique to detect, visualize and localize antigens at the cellular level. Common IHC
protocols use primary and secondary antibodies to indirectly
detect antigens in frozen or paraffin-embedded tissue sections.
Detection schemes for IHC include dye deposition for enzyme
labeled antibodies, fluorescence for fluorescent labeled
antibodies and silver enhancement for systems using goldlabeling.
What is TSA?
™

TSA (Tyramide Signal Amplification) is a powerful, patented
technology from PerkinElmer that significantly enhances both
chromogenic and fluorescent signals. It is easily integrated
into standard nonradioactive ISH or IHC protocols, provided
that horseradish peroxidase (HRP) is in the system.

5

How does TSA™ Plus Biotin Signal
Amplification work?
TSA Plus Biotin technology uses HRP to catalyze the covalentdeposition of biotin labeled amplification reagent onto tissue
sections or cell preparation surfaces that have been previously
blocked with proteins. The reaction is quick (less than 10
minutes) and results in the covalent deposition of numerous
biotin labels immediately adjacent to the immobilized HRP
enzyme. These labels can then be indirectly detected bystandard chromogenic or fluorescent techniques, withsignificant enhancement of the signal. Detection isaccomplished through the use of a streptavidin with an
enzyme or fluorophore conjugate. Because the added labels
are deposited proximal to the initial immobilized HRP enzyme
site, there is minimal loss in resolution. This signal
amplification technique may be applied to both ISH and IHC.
What ISH and IHC mediums are compatible withTSA Plus Biotin?
TSA Plus Biotin has been successfully applied to the following media: formalin-fixed/paraffin-embedded sections, cryostat sections, and cultured cells.

6B. The TSA Plus Biotin Kit
The TSA Plus Biotin Kit contains the following
components necessary for signal amplification:•

Biotin Amplification Reagent,•

1X Plus Amplification Diluent

Intended Use
The intended use of this kit is to amplify signals generated by
Horseradish Peroxidase in nonradioactive ISH and IHC
applications. The reagents in this kit have been optimized
for use in slide based assays. These kits are not suitable foruse on membranes or microtiter plates. Labeled streptavidin,
blocking reagent and other components should be purchased
separately.
FOR LABORATORY USE.

Safety Note
All reagents are classified as nonhazardous. We
strongly recommend wearing disposable gloves andsafety glasses while working. Thorough washing ofhands after handling is also recommended. Do noteat, smoke, or drink in areas in which reagents arehandled.

7

C. Components of TSA Plus Biotin Kits
NEL749A001KT

50-150 slides *Reagent

Amount1X Plus Amplification Diluent

15 mL

Biotin Amplification Reagent

1 vial

NEL749B001KT

250-750 slides *Reagent

Amount1X Plus Amplification Diluent

5x15 mL

Biotin Amplification Reagent

5 vialBiotin Amplification Reagent is supplied as a solid.
*

The number of slides is determined by the reagent
volume (approximately 100-300 uL) which is needed
to completely cover the cells or tissue section on theslide.

Storage and Stability
Upon receipt, the TSA Plus Biotin kit should be stored at 4°C.
The components in this kit are stable for 6 months underproper storage conditions. Do not use beyond expiration date8

listed on kit.
Critical Reagents Required But Not Supplied
•

•
•
•
•
•

HRP-labeled reagent, for example
o SA-HRP for use with biotin-labeled probes
or antibodies (PerkinElmer cat. no.
NEL750001EA)o HRP-labeled secondary antibody
o Anti-digoxigenin-HRP for use with DIGlabeled probes (for example Roche AntiDigoxigenin-POD cat. no. 11 207 733 910)
DMSO (molecular biology or HPLC grade)
Buffer components
Blocking reagent (PerkinElmer cat. no. FP1020)
Labeled streptavidin or anti-biotin
Chromogenic substrate as appropriate (BCIP/NBT,
DAB, AEC, etc.)

9

II.

PROTOCOL FOR ISH

A. Overview Protocol for TSA Plus Biotin for In
Situ
Hybridization
Standard non- Quench endogenous peroxidase activity (if needed)radiometricTissue permeabilization (if needed)
ISH
Probe hybridization
Post-hybridization stringency washes↓
Block slides for 30 min. in TNB buffer at room
Blocking Step
temperature↓
Incubate slides with appropriate HRP reagent (antiIncorporation
DIG-HRP, SA-HRP, etc.) for 30 min. at roomof HRP
temperature↓
Wash slides 3X for 5 min. in TNT buffer at room
TSA Plus
temperature Incubate in Biotin Amplification Reagent
Amplification
working solution for 3 to 10 min. at room temperature↓
Wash slides 3X for 5 min. in TNT buffer at room
temperature↓
Visualization
Fluorescent
Chromogenic
Incubate slides in
SA-fluorophorefor 30 min. at room
temperature↓
Wash slides 3X for 5
min. in TNT buffer atroom temperature↓
Counterstain andmount for fluorescencemicroscopy

10

Incubate slides in
SA-HRP or SA-AP
for 30 min. at room
temperature↓
Wash slides 3X for 5
min. in TNT buffer atroom temperature↓
Add appropriate
chromogen, counterstain
and mount for
microscopy

B. Suggested ISH Protocol
The following is a suggested protocol for using TSA Plus
Biotin Kit for in situ hybridization signal amplification.
TSA technology requires HRP to be an integral part of thedetection protocol. This may be accomplished via the use ofvarious hapten-labeled probe/anti-hapten-HRP conjugate
combinations such as digoxigenin-labeled probe followed byanti-DIG-HRP, biotin-labeled probe followed by SA-HRP, orwith a fluorescein-labeled probe followed by anti-fluoresceinHRP. Once HRP is introduced, the Biotin Amplification
Reagent working solution is added. Visualization is done
through the use of appropriate enzyme/chromogen
combinations. . Chromogenic substrates must be bought
separately.
First Time Users
First time nonradioactive ISH users should assess the need forvarious tissue pre-treatment conditions which may benecessary to improve penetration of reagents and/or to reduce
background. A balance must be achieved between making thetarget accessible versus causing loss of target and/or
destruction of tissue morphology. Reagent penetration may be
improved by protein digestion or detergent permeabilization
prior to probe hybridization. Common protein digestion
methods include the use of 0.005-0.1% pepsin in 0.01M HClor Proteinase K (1-10 µ g/mL) in TRIS-HCl / 0.05M EDTA.
Cell preparations are often permeabilized with detergents suchas saponin or Triton X-100. Background may be reduced

11

using procedures such as acetylation of tissue and/or inhibitionof endogenous enzyme (peroxidase or alkaline phosphatase)activity.

First time TSA Plus Biotin users should apply this to a
proven ISH system.

Controls
Always run control slides with each experiment! These should
include an unamplified control slide (i.e., include specificprobe but eliminate TSA regents) and an amplified negative
control (i.e., no probe or non-specific probe + TSA reagents)
slide. In addition to proving validity of results, control slides
are often beneficial in determining the cause of non-specificbackground.
Reagent Titration
In general, researchers have found that TSA requires lower
probe and conjugate concentrations for optimal results whencompared with standard unamplified nonradioactive methods:
1. Probe titration:
Probe concentration must be optimized. It should beassessed using the standard concentration used inunamplified nonradiometric procedures, and at reduced concentrations of 5, 10, and 20-fold - less inthe hybridization mix. In general, a 10-fold reduction

12

in probe concentration has most often been found to
be optimal. Failure to establish appropriate probe
concentration can result in little to no signal
development.

2. Titration of enzyme conjugates:
Incubation with an appropriate HRP conjugate isrequired for the activation of the Biotin amplification
reagent. Appropriate HRP conjugate concentrations
to assess include supplier’s recommended starting
concentration, 2-fold less, and 5-fold less. In cases
where no signal and no background are seen, it may
be necessary to use an increased concentration
instead. For example, if the recommended starting
titer is 1:100, run slides with HRP conjugate at 1:50,
1:100, 1:200, and 1:500.

Quenching Endogenous Peroxidase
Activation and covalent binding of the Biotin Amplification
Reagent is catalyzed by HRP. To minimize background,endogenous peroxidase activity, if present, must be quenched.
Users should establish the need for doing this and optimal
methodology specific to the tissues or cells being stained.
Options include:•••

0.3% H2O2 to 3% H2O2
Methanol or PBS as diluent for H2O2
Incubation time of 10 to 60 minutes

For paraffin-embedded tissues, quenching can be done after
dewaxing and alcohol rehydration but before the protease13

digestion step. For frozen tissue or cell preps, quenching canbe done following fixation and before the protease digestion
step. After quenching wash with TN or 1X PBS buffer for 5
minutes. See Li et al from references section for moresuggestions.
Failure to establish optimum tissue pre-treatments and reagent concentrations may result in poor signal amplification
and/or increased background.
Volumes
The protocol is written for minimal volumes of reagent (e.g.,
100-300 µL). Reagent volumes used should be sufficient tocompletely cover cells or tissue sections on slide. If larger
volumes are necessary to cover the samples, make the appropriate changes in the protocol.
Technical Support
If there are any further questions regarding TSA in your ISH
system, please contact PerkinElmer Technical Support
Department before proceeding. Refer to the Troubleshooting
Guide section for local listing. Technical assistance is alsoavailable through our web site at www.perkinelmer.com/tsa.

14

C. Standard ISH Protocol
1. Preparation of Buffers and ReagentsThe following buffers and reagents are required for slide
preparation and TSA Plus Biotin amplification.
Biotin Amplification Reagent Stock Solution
Biotin Amplification Reagent is supplied as a solid and may
be difficult to see in the vial. Reconstitute by adding 0.3
mL/vial of DMSO (dimethyl sulfoxide -molecular biology or
HPLC-grade) to make the Biotin Amplification Reagent Stock
Solution. Biotin Amplification Reagent Stock Solution, whenstored at 4° C, is stable for at least six 6 months. (Note:DMSO should freeze at 4° C. Thaw the Stock Solution, before
each use.)
Biotin Amplification Reagent Working Solution
Before each procedure, dilute the Biotin Stock Solution 1:50
using 1X Plus Amplification Diluent to make the Biotin
Working Solution. Approximately 100-300 µL of Biotin
Working Solution is required per slide. Discard any unused
portion of Biotin working solution.
Wash Buffer
TNT Wash Buffer
0.1 M TRIS-HCl, pH 7.5
0.15 M NaCl
0.05% Tween®20
Other wash buffers (such as PBS) may be used. Substitution of15

0.3% Triton X-100 for the 0.05% Tween-20 is also possible.
Users should validate the use of alternative wash buffers with
their own systems.

Blocking Buffer
TNB Blocking Buffer
0.1 M TRIS-HCl, pH 7.5
0.15 M NaCl
0.5% Blocking Reagent (available separately, catalog number
FP1020)Add Blocking Reagent slowly in small increments to buffer
while stirring. Heat gradually to 55°C with continuous stirring
to completely dissolve the Blocking Reagent. (This shouldtake no longer than 30-60 minutes. The solution will appear
milky. Bring to room temperatue befor using. Aliquot andstore at -20°C for long term use.
2. Procedural Notes
•
•
•

•
•

Do not let slides dry out between steps.
A humidified chamber is recommended for all
incubation steps (i.e., a damp paper towel in acovered box).
Drain off as much of the incubation solutions as
possible, before the addition of the next solution, toprevent reagent dilution and uneven staining. Blot
area around, but not on, tissue section using a tissue.
Be sure to use enough volume of each reagent tocompletely cover sections or cells.
Optional: Use of coverslips may reduce reagent
evaporation, especially during steps which require
long incubation at elevated temperatures (such asprobe hybridization). However care must be taken

16

upon removal to prevent damage to tissues or cells.
3. Step by Step Protocol
The following is a suggested protocol for the use of TSA
Plus Biotin in an ISH protocol.

Step 1: Slide Preparation
Prepare tissues or cells using standard fixation andembedding techniques. Dewax and rehydrate slides
according to standard procedures.

Step 2: Standard Non-radioactive ISH
Technique
Follow standard non-radioactive in situ hybridization techniques. Include tissue permeabilization (ifneeded) and quenching of endogenous peroxidase
activity (if needed). Probe hybridization (with digoxigenin, biotin, or fluorescein-labeled probes)
should be done using aconcentration determined in
optimization studies (see p. 9) followed by posthybridization stringency washes.
NOTE: Always run an unamplified control slide and an
amplified negative control slide with eachexperiment.

Step 3: Blocking Step
Incubate slides with 100-300 µL of TNB Buffer in a17humidified chamber for 30-60 minutes at room temperature or at 40C overnight.
Step 4: Introduction of HRP
Incubate slides for 30 minutes at room temperature orat 40C overnight. in a humidified chamber withappropriate HRP-labeled reagent using either:a.

DIG-labeled probes: 100-300 µL of antidigoxigenin-HRP (Roche anti-DIG-POD
Cat. No. 11 207 733 910) diluted 1:100 in
TNB Buffer,orb.

Biotin-labeled probes: 100-300 µL of SAHRP (PerkinElmer Cat. No.
NEL750001EA) diluted 1:2000 in TNB
Buffer.
orc.

Fluorescein-labeled probes: 100-300 µLof anti-fluorescein-HRP (PerkinElmer
Cat. No. NEF710001EA) diluted 1:250 in
TNB Buffer.

NOTE: HRP-labeled reagents are available from a varietyof vendors. Appropriate concentration for use
should be established as per optimization studies
suggested on p.9).
Step 5: Wash
Wash the slides 3X for 5 minutes each in18

TNT Buffer at room temperature with agitation.
Step 6: TSA Plus Biotin Amplification
Pipet 100-300 µL of the Biotin Amplification Reagent Working Solution (p. 11) onto each slide.
Incubate the slides at room temperature for 3 to 10
minutes.
Step 7: Wash
Wash the slides 3X for 5 minutes each in TNT buffer
at room temperature with agitation.

Step 8: Visualization of Deposited Biotin
Deposited biotin may be visualized for fluorescent or
chromogenic detection.
For fluorescent detection, incubate slides withfluorophore labeled streptavidin as directed by the
manufacturer.
For chromogenic detection, incubate slides with HRP
or AP labeled streptavidin followed by detection with
an appropriate chromogenic substrate.

19

III.

PROTOCOL FOR IHC

A.

Overview Protocol for TSA Plus Biotin
Immunohistochemistry
Quench endogenous peroxidase activity (if needed)↓
Block slides for 30 min. in TNB buffer at room
temperatureStandard
IHC

Incorporation of HRP

TSA Plus
Amplification↓
Incubate slides in primary antibody for 30-60 minutesat room temperature↓
Wash slides 3X for 5 min. in TNT buffer at room
temperature↓
Incubate slides inbiotinylated secondary
antibody 30-60 min. atroom temperature
Incubate slides in
Wash slides 3X for 5
HRP labeled secondary
antibody for 30 min. ator min. in TNT buffer atroom temperatureroom temperature
Incubate slides in SAHRP for 30 min. atroom temperature↓
Wash slides 3X for 5 min. in TNT buffer at room
temperature↓
Incubate in Biotin Amplification Reagent working
solution for 3 to 10 min. at room temperature↓
Wash slides 3X for 5 min. in TNT buffer at room
temperature↓

20

Visualization

Fluorescent

Chromogenic

Incubate slides in
SA-fluorophorefor 30 min. at room
temperature↓
Wash slides 3X for 5
min. in TNT buffer atroom temperature↓
Counterstain andmount for fluorescencemicroscopy

Incubate slides in
SA-HRP or SA-AP
for 30 min. at room
temperature↓
Wash slides 3X for 5
min. in TNT buffer atroom temperature↓
Add appropriate
chromogen, counterstain
and mount for
microscopy

B. Suggested IHC Protocol
The following is a suggested protocol for using TSA Plus
Biotin for immunohistochemistry signal amplification.
TSA technology requires HRP to be an integral part of thedetection protocol. This may be accomplished by using either
an HRP labeled secondary antibody or a hapten labeledsecondary antibody followed by an appropriate HRP
conjugate. Once HRP is introduced, the Biotin Amplification
Reagent Working Solution is added. Visualization is done
through the use of appropriate labeled streptavidin for
fluorescent or chromogenic detection.
First Time Users
First time users should apply TSA Biotin Plus to a proven IHC

21system.
Controls
Always run control slides with each experiment. Include atleast one negative control slide (eliminating primary antibody
but including the TSA Plus reagents) and one unamplified
control slide (include all reagents except TSA-Plus). In
addition to proving validity of results, control slides may bebeneficial in determining the cause of non-specific background.

Reagent Titration
Failure to establish optimum reagent concentrations may
result in poor amplification and/or increased background.
Primary and/or secondary antibody dilutions should be optimized when applying TSA for the first time. The following
test slides are recommended:

Test slide 1:
Primary or Secondary Ab at manufacturer's recommended
dilution.
Test slide 2:
5 fold dilution of slide #1 Ab concentration.
Test slide 3:
5 fold dilution of slide #2 Ab concentration.
Test slide 4:
5 fold dilution of slide #3 Ab concentration.

22

Test slide 5:
Unamplified control.
More than the above dilutions may be necessary. In cases
where low signal is obtained, increasing the dilution of the
primary antibody often leads to better signal amplification.
In many cases, the optimal dilution of the primary antibody
for TSA detection is 1000-fold less than that used forstandard detection.

Quenching Endogenous Peroxidase
Activation and covalent binding of the Biotin Amplification
Reagent is catalyzed by HRP. To minimize background,endogenous peroxidase activity, if present, must be quenched
before the immunostaining protocol. Users should establish
the need for doing this and optimal methodology specific to
the tissues or cells being stained.
Options include:•••

0.3% H2O2 to 3% H2O2
Methanol or PBS as diluent for H2O2
Incubation time of 10 to 60 minutes

For paraffin-embedded tissues quenching can be done after
dewaxing and alcohol rehydration but before the blocking
step. For frozen tissue or cell preps, quenching can be done
following fixation and before the blocking step. See Li et al
from the references for more suggestions.
After quenching wash with TNT buffer for 5 minutes.

23

Volumes
The protocol is written for minimal volumes of reagent (e.g.,
100-300 µL). Reagent volumes used should be sufficient tocompletely cover cells or tissue sections on slide. If larger
volumes are necessary to cover the samples, make the appropriate changes in the protocol.
Technical Support
If there are any further questions regarding TSA in your IHC
system, please contact PerkinElmer Technical Support Department before proceeding. Refer to the Troubleshooting
Guide section for local listing. Technical assistance is alsoavailable through our web site at www.perkinelmer.com/tsa.

C. Standard IHC Protocol
1. Preparation of Buffers and ReagentsThe following buffers and reagents are required for slide
preparation and TSA Plus Biotin amplification.
Biotin Amplification Reagent Stock Solution
Biotin Amplification Reagent is supplied as a solid. Reconstitute by adding 0.3 mL/vial of dimethyl sulfoxide (DMSOmolecular biology or HPLC-grade) to make the Biotin
Amplification Reagent Stock Solution. Biotin Amplification
Reagent Stock Solution, when stored at 4° C, is stable for atleast six 6 months. (Note: DMSO should freeze at 4° C.
Thaw the Stock Solution before each use.)24

Biotin Amplification Reagent Working Solution
Before each procedure, dilute the Biotin Amplification
Reagent Stock Solution 1:50 using 1X Plus Amplification
Diluent to make the Biotin Amplification Working Solution.
Approximately 100-300 µL of Biotin Amplification Working
Solution is required per slide. Discard any unused portion of
Biotin working solution.
Wash Buffer
TNT Wash Buffer
0.1 M TRIS-HCl, pH 7.5
0.15 M NaCl®

0.05% Tween 20
Other wash buffers such as PBS may be used. Substitution of
0.3% Triton X-100 for the
0.05% Tween-20 is also possible. Users should validate
the use of wash buffer alternatives with their own systems.

NOTE: When staining cell surface/membrane targets, do
NOT include detergent in wash buffer or diluents.
Detergents may cause stripping or alteration of cell surface
antigens.
Blocking Buffer
TNB Blocking Buffer
0.1 M TRIS-HCl, pH 7.5

25

0.15 M NaCl
0.5% Blocking Reagent (available separately, catalog number
FP1020)Add Blocking Reagent slowly in small increments to buffer
while stirring. Heat gradually to 55°C with continuous stirring
to completely dissolve the Blocking Reagent. (This shouldtake no longer than 30-60 minutes. The solution will lookmilky. . Bring to room temperature before using.
Aliquot and store at -20°C for long term use.

2. Procedural Notes
•
•
•

•
•

Do not let slides dry out between steps.
A humidified chamber is recommended for all
incubation steps (i.e., a damp paper towel in acovered box).
Drain off as much of the incubation solutions as
possible, before the addition of the next solution, toprevent dilution and uneven staining. Blot areaaround, but not on, tissue section using a tissue.
Be sure to use enough volume of solutions to coversections or cells.
Optional: Use of coverslips may reduce reagent
evaporation. However care must be taken upon
removal to prevent damage to tissues or cells.

3. Step by Step Protocol
The following is a suggested protocol for the use of TSA
Plus Biotin in IHC applications.

26

Step 1: Slide Preparation
Prepare tissues or cells for detection with TSA Plus Biotin
using standard fixation and embedding techniques. Dewax andrehydrate using standard protocols. Quench endogenous
peroxidase activity if necessary.
NOTE: Always run an unamplified control slide and an
amplified negative control slide with eachexperiment.

Step 2: Blocking Step
Incubate slides with 100-300 µL of TNB Buffer in ahumidified chamber for 30-60 minutes at room temperature orat 40C overnight. (Note: PBS may be substituted for the TN
buffer.)
Step 3: Primary Antibody Incubation
Drain off the TNB Buffer and apply 100-300 µL of the
primary antibody, diluted in TNB Buffer. Incubate theprimary antibody preparation per the manufacturer's
instructions regarding incubation time and temperature
requirements. Use concentration determined in optimization
studies (see p.19).

Step 4: Wash
Wash the slides 3X for 5 minutes each in
TNT Buffer at room temperature with agitation.

27

Step 5: Introduction of HRP
Incubate slides with HRP by doing one of the following:a)100-300 µL of HRP labeled secondary antibody
diluted in TNB Buffer.

orb)100-300 µL of biotinylated secondary antibody
diluted in TNB Buffer. Incubate 30-60 minutes in ahumidified chamber. Wash the slides for 3 X 5
minutes TNT buffer at room temperature withagitation. Follow by 100-300 µL of SA-HRP dilutedin TNB Buffer. Use SA-HRP at 1:2000 if using
PerkinElmer Cat. # NEL750001EA. When using
alternative suppliers, reagents should be optimized
for use with TSA starting with manufacturer's
recommended dilutions. Incubate slides in ahumidified chamber for 30-60 minutes at room
temperature or at 40C overnight.

Step 6: Wash
Wash the slides 3X for 5 minutes each in TNT Buffer at room
temperature with agitation.

Step 7: TSA Plus Biotin Amplification
Pipette 100-300 µL of the Biotin Amplification Working
Solution (p. 21) onto each slide. Incubate the slides at room28

temperature for 3 to 10 minutes.
Step 8: Wash
Wash the slides 3X for 5 minutes each inTNT Buffer at room
temperature with agitation.

Step 9: Visualization of Deposited Biotin
Deposited biotin may be visualized with fluorescent or
chromogenic detection.
For fluorescent detection, incubate slides with fluorophorelabeled streptavidin as directed by the manufacturer.
For chromogenic detection, incubate slides with HRP or AP
labeled streptavidin followed by detection with an appropriate
chromogenic substrate.

29

IV. TROUBLESHOOTING GUIDEA. In Situ Hybridization (ISH)

PROBLEM

REMEDY

Low Signal•••

Excess Signal

•
•
•

High
Background

•
•
•
•
•
•
•
•

Titer HRP conjugate used forvisualization to determine optimum
concentration for signal amplification.
Increase concentration of Biotin
Amplification Reagent solution and/or
lengthen incubation time.
Add tissue permeabilization step tofacilitate penetration of reagents.
Decrease concentration of HRP
conjugate introduced prior toamplification.
Decrease probe concentration.
Decrease Biotin Amplification Reagent
incubation time. Decrease concentrationof streptavidin-enzyme conjugate usedfor visualization.
Decrease concentration of HRP
conjugate.
Decrease probe concentration.
Shorten chromogenic development time.
Lengthen endogenous peroxide
quenching step.
Samples may contain endogenous biotin.
Switch to fluorescein or digoxigeninlabeled probes.
Filter buffers.
Increase number and/or length of
washes.
Nonqualified or contaminated blocking
reagent used. Use PerkinElmer Blocking
Reagent (FP1020).

30

B. Immunohistochemistry (IHC)

PROBLEM

REMEDY

Low Signal•••

Excess Signal

•
•
•

High
Background

•
•
•
•
•

Titer primary and/or secondary
antibodies to determine optimum
concentration for signal amplification
Increase concentration of Biotin
Amplification Reagent solution and/or
increase incubation time.
In some cases it may be necessary toutilize antigen retrieval techniques tounmask the target.
Decrease concentration of primary
and/or secondary antibody or HRP
conjugates.
Decrease Biotin Amplification Reagent
incubation time.
Decrease concentration of Biotin
conjugates used for visualization.
Filter buffers
Decrease concentration of primary
and/or secondary antibody or HRP
conjugates.
Lengthen endogenous peroxide
quenching step.
Increase number and/or length of
washes. Shorten chromogenic
development time.
Nonqualified or contaminated blocking
reagent used. Use PerkinElmer Blocking
Reagent (FP1020).

31

C. Customer Technical Support Services

For Further Technical Information, or, toPlace an Order Contact:
Web site: http://www.perkinelmer.com/
Email: TechSupport@perkinelmer.com
In the U.S.:

PerkinElmer
Technical Support
Department at (800)762-4000.

Outside the U.S.:

Contact your local
PerkinElmer sales
office or distributor.

32

33

V. Ordering information
TSA Plus Kits: 10 to 20 times more sensitive than regular TSA (includes
labeled amplification reagent and amplification diluent )Product

No. of Slides

Product No.

50–150

NEL741001KT

250–750

NEL741B001KT

50–150

NEL742001KT

250–750

NEL742B001KT

50–150

NEL744001KT

250–750
50–150

NEL744B001KT

250–750

NEL745B001KT

TSA Plus Cyanine 3/Cyanine 5 Kit

50–150

NEL752001KT

TSA Plus Cyanine 3/Fluorescein Kit

50–150

NEL753001KT

TSA Plus Cyanine 5/Fluorescein Kit

50–150

NEL754001KT

TSA Plus Fluorescein/TMR Kit

50–150

NEL756001KT

TSA Plus Fluorescence Palette Kit(contains one each of Fluorescein,
TMR, Cyanine 3 and Cyanine 5)

10–35

NEL760001KT

50–150

NEL749A001KT

250–750

NEL749B001KT

TSA Plus Fluorescence Kits
TSA Plus Fluorescein Kit
TSA Plus TMR Kit
TSA Plus Cyanine 3 Kit
TSA Plus Cyanine 5 Kit

NEL745001KT

TSA Plus Multi‐Fluor Combination Kits

TSA Plus Biotin Kits
TSA Plus Biotin Kit

34

TSA Plus DNP Systems: sensitive, biotin‐free chromogenic detection (includes
DNP amplifcation reagent , amplification diluent, anti‐DNP enzyme conjugate
and blocking reagent)
Product

TSA Plus DNP (AP) System
TSA Plus DNP (HRP) System

No. of Slides

Product No.

25–75

NEL746B001KT

50–15025–75

NEL746A001KT
NEL747B001KT

50–150
NEL747A001KT
TSA Fluorescence Systems (includes labeled amplification reagent,
amplification diluent, streptavidin HRP and blocking reagent)
Product
No. of Slides
Product No.
50–150

NEL701A001KT
NEL701001KT

TSA TMR System

100–300
100–300

NEL702001001KTTSA Coumarin System

100–300

NEL703001KTTSA Cyanine 3 System
TSA Cyanine 5 System

50–15050–150

NEL704A001KT

TSA Fluorescein System

NEL705A001KT
TSA Biotin Systems (includes labeled amplification reagent, amplification
diluent, streptavidin HRP and blocking reagent)
50–150
NEL700A001KT
TSA Biotin System
200–600
NEL700001KT

35

TSA Reagent Packs (includes labeled tyramide and amplification diluent)
Product

No. of Slides

Product No.

200–600

SAT700001EATSA Biotin Tyramide Reagent Pack

1,000–3,000

SAT700B001EATSA Fluorescein Tyramide Reagent
Pack

100–300

SAT701001EA

500–1,500

SAT701B001EATSA TMR Tyramide Reagent Pack

100–300

SAT702001EATSA Cyanine 3 Tyramide Reagent
Pack

50–150

SAT704A001EA

250–750

SAT704B001EATSA Cyanine 5 Tyramide Reagent
Pack

50–150

SAT705A001EA

Complementary Products
TSA Blocking Reagent
Horseradish Peroxidase Reagents

FP1020Anti‐rabbit IgG (goat) HRP

NEF812001EA

Anti‐mouse IgG (goat) HRP

NEF822001EA

Anti‐human IgG (goat)* HRP

NEF802001EA

Anti‐DNP‐HRP

FP1129Antifluorescein‐HRP

NEF710001EAStreptavidin‐HRP
Biotin Conjugates

NEL750001EA

Anti‐rabbit IgG (goat) biotin

NEF813001EA

Anti‐mouse IgG (goat) biotin

NEF823001EA

Anti‐human IgG (goat) biotin

NEF803001EA

36

Labeled Streptavidin
Streptavidin Fluorescein

NEL720001EAStreptavidin Texas Red®

NEL721001EAStreptavidin Coumarin

NEL722001EAStreptavidin‐HRP

NEL750001EAStreptavidin‐AP
Chromogens

NEL751001EA

BCIP/NBT Substrate (For detectionof Alkaline Phosphatase)

NEL937001PKDAB Substrate (For detection ofHorseradish Peroxidase)
Alkaline Phosphatase Reagents

NEL938001EA

Anti‐Mouse IgG (Goat), AP‐Labeled

NEF814001EA

Anti‐Rabbit IgG (Goat), AP‐Labeled

NEF824001EAStreptavidin‐ AP Conjugate

NEL751001EA

Antifluorescein‐AP Conjugate

NEF709001PK

Anti‐DNP‐AP

FP113137

Hapten Labeled Deoxynucleotides (for labeling of ISH probes)
3‐Amino‐3‐Deoxydigoxigenin‐9‐dCTP

NEL562001EA

Biotin‐11‐dATP

NEL540001EA

Biotin‐11‐dCTP

NEL538001EA

Biotin‐11‐dGTP

NEL541001EA

Biotin‐11‐dUTP

NEL539001EA

DNP‐11‐dUTP

NEL551001EA

Fluorescein‐12‐dATP

NEL465001EA

Fluorescein‐12‐dCTP

NEL424001EA

Fluorescein‐12‐dGTP

NEL429001EA

Fluorescein‐12‐dUTP
NEL413001EA
Hapten Labeled Ribonucleotides (for labeling of ISH probes)
Biotin‐11‐ATP

NEL544001EA

Biotin‐11‐CTP

NEL542001EA

Biotin‐11‐GTP

NEL545001EA

Biotin‐11‐UTP

NEL543001EA

Fluorescein‐12‐ATP

NEL439001EA

Fluorescein‐12‐CTP

NEL434001EA

Fluorescein‐12‐GTP

NEL496001EA

Fluorescein‐12‐UTP

NEL414001EA

38

V. SELECTED REFERENCES
A quantitative evaluation of peroxidase inhibitors fortyramide signal amplification mediated cytochemistry
and histochemistry
G. Li, S. Amin, N. N. Okuhama, G. Liao, L. A. Mingle

Histochem Cell Biol (2006)
http://www.pubmedcentral.nih.gov/articlerender.fcgi?arti
d=1713225
Dual Fluorescent In Situ Hybridization and
Immunohistochemical Detection with Tyramide
Signal Amplification
Aliya U. Zaidi, Hideki Enomoto, Jeffrey Milbrandt, and Kevin A. RothJ Histochem Cytochem. (2000)
http://www.ncbi.nlm.nih.gov/pubmed/10990490
Zebrafish Whole Mount High-Resolution Double
Fluorescent In Situ Hybridization,
Tim Brend, Scott A. Holley

J Vis Exp. (2009)http://www.ncbi.nlm.nih.gov/pubmed/19322135
Genomic Anatomy of the Hippocampus
Carol L. Thompson, Sayan D. Pathak, Andreas Jeromin, Lydia L. Ng,
Cameron R. MacPherson, Marty T. Mortrud, Allison Cusick, Zackery L.
Riley, Susan M. Sunkin, Amy Bernard, Ralph B. Puchalski, Fred H.
Gage, Allan R. Jones, Vladimir B. Bajic, Michael J. Hawrylycz and Ed
S. LeinNeuron. (2008)
http://www.ncbi.nlm.nih.gov/sites/entrez/19109908?dopt=Abstract&holding=f1000,f1000m,isrctn
Detection of microRNAs in frozen tissue sections byfluorescence in situ hybridization using locked nucleic
acid probes and tyramide signal amplification
Asli N Silahtaroglu, Dorrit Nolting, Lars Dyrskjøt, Eugene Berezikov,
Morten Møller, Niels Tommerup & Sakari Kauppinen

Nat Protoc. (2007)
http://www.ncbi.nlm.nih.gov/pubmed/17947994

39

Introduction of Tyramide Signal Amplification (TSA)
to Pre-embedding Nanogold–Silver Staining at theElectron Microscopic Level
Seung-won Lee, Song Eun Lee, Seong Hyuk Ko, Eun Kyoung Hong,
Kwang Il Nam, Kei-ichiro Nakamura, Shuhei Imayama, Yeong-Joon
Park, Kyu Youn Ahn, Choon Sang Bae, Baik Yoon Kim, and Sung Sik
Park

J Histochem Cytochem. (2005)

http://www.ncbi.nlm.nih.gov/pubmed/15684338

40

Patents
TSA and its use are protected under U.S. Patents 5, 196, 306, 5,583,001,
5,731,158, 5,863,748, 6,372,937 and patents pending, and foreign
equivalents thereof.

For Research Use Only This product is distributed and soldfor research purposes only by the end-user in the researchmarket, and, to that extent, by purchasing this product theend-user is granted a limited license to use this product for
research only. This product is not intended for diagnostic
or therapeutic use and no license or right is granted to use
this product for diagnostic or therapeutic purposes.
Purchase does not in-clude or carry any right or license touse develop or otherwise exploit this product commercially.
Any commercial use, development or exploitation of thisproduct without the express prior written authorization of
PerkinElmer is strictly prohib-ited and constitutes aninfringement of the intellectual property rights of PerkinElmer under the aforementioned patents.

Trademarks®

TSA ™ is a trademark of PerkinElmer. TWEEN 20 is a registeredtrademark of ICI Americas, Inc. Texas Red ™ is a registered trademarkof Molecular Probes, Inc.

41

42

PerkinElmer, Inc.
940 Winter Street
Waltham, MA 02451 USA(800) 762-4000
or (+1) 203-925-4602
www.perkinelmer.com
For a complete listing of our global offices, visit
www.perkinelmer.com/contactus

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