Thermo Fisher Scientific Tyramide SuperBoost EverRed/EverBlue HRP Kits User guide

Brand
Thermo Fisher Scientific
Model
Tyramide SuperBoost EverRed/EverBlue HRP Kits
Type
User guide
For Research Use Only. Not for use in diagnostic procedures.
Tyramide SuperBoost™ EverRed/EverBlue
HRP Kits
USER GUIDE
Publication Number MAN0029784
Revision A.0
Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, California 92008 USA
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
Revision history:MAN0029784 A.0 (English)
Revision Date Description
A.0 16 October 2023 New document for Tyramide SuperBoost™ EverRed/EverBlue HRP Kits.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these
products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2023 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■CHAPTER1Productinformation .................................................. 5
Productdescription ............................................................. 5
Contents and storage ............................................................ 9
Required materials notsupplied ................................................. 11
Workflow ..................................................................... 12
■CHAPTER2Methods ............................................................. 13
Proceduralguidelines ........................................................... 13
Before youbegin ............................................................... 13
Prepare PBS-T washbuer ................................................. 13
Preparetissues ............................................................ 13
Perform peroxidaselabeling ..................................................... 14
Perform tyramidelabeling ....................................................... 15
Multiplex with primary antibodies from dierentspecies ............................ 16
Counterstain, mount, then detect thesignal ....................................... 16
■APPENDIXATroubleshooting .................................................... 17
■APPENDIXBSignaloptimization ................................................ 18
Optimize the amount of primary antibody or probe ................................. 18
Optimize the incubation time for tyramidelabeling .................................. 18
■APPENDIXCOrderinginformation .............................................. 19
SuperBoost™ reagents available for purchase separately ............................ 19
■APPENDIXDSafety ............................................................... 20
Chemicalsafety ................................................................ 20
Biological hazardsafety ......................................................... 21
Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide 3
■APPENDIXEDocumentation and support ...................................... 22
Customer and technical support ................................................. 22
Limited product warranty ........................................................ 22
Contents
4Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide
Product information
IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
Product description
Tyramide SuperBoost™ signal amplification is a highly sensitive method for the detection of targets in
multiplexable colorimetric immunohistochemistry (IHC) experiments. Tyramide SuperBoost™ technology
combines the permanence of poly-HRP-mediated tyramide signal deposition with the bright colors of
SuperBoost™ EverRed/EverBlue reagents to discern signal from noise, yielding precision and sensitivity.
Colorimetric signal anchoring in the Tyramide SuperBoost™ EverRed/EverBlue HRP Kits uses the
catalytic activity of horseradish peroxidase (HRP) for high-density tyramide labeling of a target protein,
resulting in permanent staining that is wash resistant, water/organic solvent mountable, and archivable.
The kits are easy to use and can be easily adapted to standard IHC experimental protocols using
any tissue type. Tissue labeled using a Tyramide SuperBoost™ kit can be imaged using any brightfield
microscope to produce high-resolution images. kits enable the following:
•Colorimetric and fluorescence detection and confirmation
•Co-labeling with common counterstains
•Multiplexing with DAB
13
2
Figure1 kits produce intense stable staining similar to DAB, but in multiple colors.
Formalin-fixed paran-embedded (FFPE)-preserved human tonsil tissue was processed for immunohistochemistry and
labeled with rabbit anti-Ki67 primary antibody, which was detected with the SuperBoost™ (1) EverRed Goat anti-Rabbit
IgG (Cat. No. E40967), (2) EverBlue Goat anti-Rabbit IgG (Cat. No. E40968), or (3) DAB using SuperBoost™ Goat anti-
Rabbit Poly HRP IgG (Cat. No. B40962). Images were taken using an EVOS™ M7000 Imaging System (Cat. No. AMF7000)
with 4x objective.
1
Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide 5
1234
Figure2 SuperBoost™ EverRed/EverBlue kits produce solvent resistant tissue staining.
Cryo-preserved mouse brain tissue was processed for immunohistochemistry and labeled with rabbit anti-NeuN primary
antibody, which was detected with the Tyramide SuperBoost™ EverRed Goat anti-Rabbit IgG (Cat. No. E40967) or
EverBlue Goat anti-Rabbit IgG (Cat. No. E40968). The tissue was then dehydrated and cleared using (1) the typical
workflow, (2) an additional extended 70% ethanol wash, and (3)(4) an additional extended 100% xylene wash. Images
were taken with an EVOS™ M7000 Imaging System (Cat. No. AMF7000) 10x objective using the same exposure and gain.
1
2
3
4
Figure3SuperBoost™ EverRed/EverBlue stain does not degrade over time.
FFPE-preserved human colon tissue was processed for immunohistochemistry and labeled with mouse anti-CDH17
primary antibody, which was detected with the Tyramide SuperBoost™ (1) EverRed Goat anti-Mouse IgG (Cat. No.
E40965) or (3) EverBlue Goat anti-Mouse IgG (Cat. No. E40966). Within 24 hours of staining, images were taken with an
EVOS™ M7000 Imaging System (Cat. No. AMF7000) using a 20x objective. The slides were then placed in a 45°C oven for
13 weeks to simulate aging at room temperature for a year. The slides were then reimaged (2, EverRed)(4, EverBlue) .
Chapter1Product information
Product description
1
6Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide
12
46
5
3
Figure4SuperBoost™ EverRed/EverBlue staining can be detected via fluorescence as well as brightfield
microscopy.
FFPE-preserved human breast tissue was processed for immunohistochemistry and labeled with anti-ESR1 primary
antibody, which was detected with the SuperBoost™ (1,2,3) EverRed Goat anti-Rabbit IgG (Cat. No. E40967) or (4,5,6)
EverBlue Goat anti-Rabbit IgG (Cat. No. E40968). Images were taken with an EVOS™ M7000 Imaging System (Cat. No.
AMF7000) 40x objective using (1,4) brightfield, (2,5) an EVOS™ Light Cube, RFP 2.0 (Cat. No. AMEP4952), or (3,6) an
EVOS™ Light Cube, Cy5 2.0 (Cat. No. AMEP4956) using the same exposure and gain.
12
Figure5SuperBoost™ EverRed/EverBlue kits can be used with common immunohistochemical
counterstains.
FFPE-preserved human tonsil tissue was processed for immunohistochemistry and labeled with anti-Ki67 primary
antibody, which was detected with the SuperBoost™ (1) EverRed Goat anti-Rabbit IgG (Cat. No. E40967) or (2) EverBlue
Goat anti-Rabbit IgG (Cat. No. E40968). Sample A was counterstained with hematoxylin. Sample B was counterstained
with eosin Y. Images were taken with an EVOS™ M7000 Imaging System (Cat. No. AMF7000) with a 20x objective.
Table1Counterstain compatibility when used with EverRed or EverBlue kits.
Counterstain EverRed kit EverBlue kit
Eosin Incompatible Recommended
Hematoxylin Recommended Incompatible
Methyl Green Acceptable (discolors stain) Acceptable
Nuclear Fast Red Incompatible Incompatible
Chapter1Product information
Product description 1
Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide 7
12
Figure6Multiplex protocol: Sequential staining with SuperBoost™ EverRed/EverBlue kits and DAB.
Cryo-preserved human tonsil tissue was processed for immunohistochemistry and labeled with rabbit anti-Ki67 primary
antibody and mouse anti-KRT15 primary antibody, which was detected with DAB (ImmPACT DAB kit, OEM from vector)
using HRP-goat anti-rabbit IgG and then with the SuperBoost™ (1) EverRed Goat anti-Mouse IgG (Cat. No. E40965), or (2)
EverBlue Goat anti-Mouse IgG (Cat. No. E40966). Images were taken using an EVOS™ M7000 Imaging System (Cat. No.
AMF7000) with 20x objective.
1
4115
23
Figure7Multiplex protocol: Sequential staining with SuperBoost™ EverRed/EverBlue kits can be
detected via fluorescence.
Cryo-preserved mouse intestine tissue was processed for immunohistochemistry, stained with DAPI and labeled with
rabbit anti-Ki67 primary antibody and mouse anti-CDH17 antibody, which was detected with the (1) SuperBoost™
EverRed Goat anti-Rabbit IgG (Cat. No. E40965) and EverBlue Goat anti-Mouse IgG (Cat. No. E40966). Images were
taken with an EVOS™ M7000 Imaging System (Cat. No. AMF7000) 20x objective using (5) brightfield, (3)an EVOS™ Light
Cube, RFP 2.0 (Cat. No. AMEP4952), (4) an EVOS™ Light Cube, Cy5 2.0 (Cat. No. AMEP4956), or (2) an EVOS™ Light
Cube, DAPI 2.0 (Cat. No. AMEP4950).
Chapter1Product information
Product description
1
8Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide
4x
20x
4x
Figure8 Multiplex protocol: Sequential staining with SuperBoost™ EverRed/EverBlue kits.
Cryo-preserved mouse intestine tissue was processed for immunohistochemistry and labeled with rabbit anti-Ki67 primary
antibody and mouse anti-CDH17 primary antibody, which were detected with the SuperBoost™ EverRed Goat anti-Rabbit
IgG (Cat. No. E40965) and then with EverBlue Goat anti-Mouse IgG (Cat. No. E40966). Images were taken using an
EVOS™ M7000 Imaging System (Cat. No. AMF7000) with a 4x or 20x objective.
Contents and storage
Table2Tyramide SuperBoost™ EverRed/EverBlue HRP Substrate kits standard kit components.
Item Amount[1] Concentration Storage[2]
1X Endogenous peroxide quencher (stabilized 3%
H2O2 solution)
(Component A)
28.5mL 1X
•2–8°C
•Desiccate
•Protect from light
•Do not freeze
1X Blocking Buer (10% goat serum)
(Component B)
22.5mL 1X
Poly-HRP-conjugated secondary antibody/HRP-
conjugated streptavidin
(Component C)
22.5mL
(150 slides)
1X
10X Tyramide HRP substrate
(Component D1)
3mL 10X
2X Hydrogen peroxide
(Component D2)
20mL 2X
2X HRP reaction buer
(Component D3)
25mL 2X
Chapter1Product information
Contents and storage 1
Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide 9
Table 2 Tyramide SuperBoost EverRed/EverBlue HRP Substrate kits standard kit
components.(continued)
Item Amount[1] Concentration Storage[2]
SuperBoost™ EverRed/EverBlue reagent
(Component E1)
3 vials — •2–8°C
•Desiccate
•Protect from light
•Do not freeze
SuperBoost™ EverRed/EverBlue development buer
(Component E2)
200µL —
[1] Sufficient material is provided for 150 slides based on the protocols. See Table4 for more information about individual Tyramide
SuperBoost™ kits and stand-alone reagents.
[2] When stored as directed, the product is stable for 6 months after receipt.
Table3Available Tyramide SuperBoost™ EverRed/EverBlue HRP Substrate kits.
Labeled tyramide
Tyramide conjugate
Kit size[1]
Standard tyramide
conjugate (stand-
alone)[2]
Goat anti-
mouse IgG[3]
Goat anti-
rabbit IgG[3] Streptavidin
SuperBoost™ EverRed
HRP Kit E40963 E40965 E40967 E40969
150 slides
SuperBoost™ EverBlue
HRP Kit B40964 E40966 B40968 B40970
[1] Sufficient material is provided for up to 150 (based on catalog number) 18-mm x 18-mm coverslips using 100 μL of solution per slide
in most critical incubation steps. This volume can be adjusted for different size samples.
[2] Tyramide reagents provided in each kit can be purchased as stand-alone reagents.
[3] Poly-HRP-conjugated secondary antibody (Component B) provided in each kit can be purchased as stand-alone reagent.
Table4Tyramide SuperBoost™ EverRed/EverBlue HRP Substrate combination kits.
Item Catalog No. Kit size
Tyramide SuperBoost™ EverRed HRP Substrate Kit E40971
5 x 150 slides[1]
Tyramide SuperBoost™ EverBlue HRP Substrate Kit E40972
[1] Sufficient material is provided for up to 750 (based on catalog number) 18-mm x 18-mm coverslips using 100 μL per slide in most
critical incubation steps. This volume can be adjusted for different size samples.
Chapter1Product information
Contents and storage
1
10 Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide
Required materials not supplied
Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that
the material is available from fisherscientific.com or another major laboratory supplier.
Catalog numbers that appear as links open the web pages for those products.
Item Source
Tissue (use positive and negative controls when needed) MLS
Slides, coverslips, or containers MLS
Conjugated probes for FISH MLS
Primary or secondary antibodies[1] MLS
PBS (phosphate buered saline), pH 7.4
(without calcium, magnesium, or phenol red in solution)
10010031
95% ethanol MLS
Distilled water, ultrapure 15230-147
Hydrophobic barrier pen R3777
Image-iT™ Fixation/Permeabilization Kit R37602
Image-iT™ 4% Formaldehyde Fixative Solution in PBS (methanol-free) FB002
Image-iT™ Paraformaldehyde 4% Fixative Solution in 0.1 M Phosphate Buer (methanol-free) I28800
Antibody Diluent 003118
Endogenous Biotin-Blocking Kit E21390
Citrate buer, pH 6.0, concentrate 005000
Epredia™ Cytoseal™ 60 Mountant 8310-4
ProLong™ Glass Antifade Mountant P36982
[1] To search the Thermo Fisher Scientific primary antibody collection, visit the antibody search tool at Thermo Fisher Scientific
antibody search tool.
Chapter1Product information
Required materials not supplied 1
Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide 11
Workflow
Use Tyramide SuperBoost™ EverRed/EverBlue HRP kits workflow
Fix, permabilize, and block tissue (page13)
Apply primary antibody (page14)
Apply poly-HRP-conjugated secondary antibody or HRP-conjugated
streptavidin (page14)
Apply SuperBoost™ tyramide reagent in reaction buer (page15)
Develop color using EverRed/EverBlue reagent (page15)
Detect signal (page16)
Chapter1Product information
Workflow
1
12 Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide
Methods
Procedural guidelines
IMPORTANT! After resuspension and use, store Tyramide SuperBoost™ EverRed/EverBlue solutions in
the dark at 4°C. Prolonged contact with heat and light degrades the reagents and can cause decreased
staining intensity.
•Although the protocols for general IHC tissue processing can be similar, there is no universal
protocol. Each laboratory will use slightly dierent preparation methods for their tissues. Tissue
processing can be one of the most important parameters aecting staining results, therefore
dierent tissue types & preparations may require dierent treatments for optimum results. When
using the Tyramide SuperBoost™ kits for the first time, optimize the protocols following the
guidelines in AppendixB, “Signal optimization”.
•A hydrophobic barrier pen (wax pen) can be used to hold liquid reagents on the sample slide or
coverslip.
•Do not let the tissue samples dry out.
•For longer incubations, a humidified chamber (e.g., a covered box with a damp paper towel) can be
used.
Before you begin
Prepare PBS-T wash buer
•Prepare 1 L of 1X PBS-T ( PBS with 0.05% Tween™-20 Detergent) by adding 0.5mL of Tween™-20
Detergent in 1L of 1X PBS and mix well.
•Prepare 10mL of 0.1% Triton™ X-100 Detergent in 1X PBS.
Prepare tissues
The Tyramide SuperBoost™ system is compatible with all tissue types that are labeled using standard
IHC techniques.
•For FFPE tissues: Deparanize, rehydrate, and antigen-retrieve the tissue according to standard
IHC protocols before treating the tissue for endogenous peroxidase activity in step1 on page14.
Note: Appropriate antigen retrieval is important for obtaining optimal staining results.
•For cryo-preserved tissues: Fix the tissue according to standard IHC protocols before treating it for
endogenous peroxidase activity in step1 on page14.
2
Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide 13
Perform peroxidase labeling
Appropriate antigen retrieval is important for obtaining optimal staining results.
1. Quench the endogenous peroxidase activity of the sample by adding sucient drops of 1X
endogenous peroxide quencher (stabilized 3% H2O2 solution) (Component A) to cover the sample
and incubate for 10 minutes at room temperature. For tissues with more endogenous peroxidase
activity, incubation times may need to be extended and optimized.
2. Rinse the tissue 3 times with 1X PBS-T at room temperature.
3. (Optional) If using HRP-conjugated streptavidin, block endogenous biotin in the sample with the
Endogenous Biotin-Blocking Kit (Cat. No. E21390). Rinse the tissue 3 times with 1X PBS-T at room
temperature before proceeding to the next step.
4. Add 2–3 drops (approximately 100–150µL) of 1X Blocking Buer (10% goat serum) (Component B)
to the sample and incubate for 30 minutes at room temperature.
IMPORTANT! After resuspension and use, store EverRed/EverBlue solutions in the dark at 4°C.
Prolonged contact with heat and light degrades the reagents and can cause decreased staining
intensity.
5. Label the tissue with primary antibody (mouse or rabbit host).
If using a SuperBoost™ kit with streptavidin, use a biotin-conjugated primary antibody or other
ligand. Dilute the antibody or biotin-conjugated ligand in 1X Blocking Buer (10% goat serum)
or another compatible blocking solution (for example, 2% BSA or BlockAid™ Blocking Solution)
(Cat. No.B10710) and incubate with the tissue for 60 minutes at room temperature or overnight at
2–8°C.
6. Rinse the tissue for 10 minutes with 1X PBS-T at room temperature. Repeat the rinse 3 times.
7. Add 2–3 drops (approximately 100–150 µL) of poly-HRP-conjugated secondary antibody or
HRP-conjugated streptavidin (Component C) to the tissue and incubate for 60 minutes at room
temperature or overnight at 2–8°C.
Note: If you observe nonspecific signal, shorten the incubation period.
8. Rinse the tissue in 1X PBS-T for 10 minutes at room temperature. Repeat the rinse 3times.
Chapter2Methods
Perform peroxidase labeling
2
14 Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide
Perform tyramide labeling
1. Prepare a tyramide working solution according to Table5.
IMPORTANT! Use working solution within 1 hour of preparation. Do not store excess working
solution after that time.
Table5Tyramide working solution component volumes.
Component
Number of coverslips (18mm x 18mm)
1 5 10 50 100
10X Tyramide HRP substrate 20µL 100µL 200µL 1mL 2mL
2X H2O2 for reaction buer 90µL 450µL 900µL 4.5mL 9mL
2X HRP Reaction Buer 90µL 450mL 900mL 4.5mL 9mL
Note: The volumes in this table are based on 200 µL of tyramide working solution needed per
18-mm x 18-mm coverslip. This volume can be adjusted based on the size of the coverslip or the
volume needed per well in a microplate.
2. Apply 200 μL of tyramide working solution to the tissue and incubate for 10 minutes at room
temperature.
3. Rinse the tissue with 1X PBS-T 3 times for 5 minutes each time.
4. Resuspend the EverRed or EverBlue reagent in 1.5 mL of distilled water.
5. Centrifuge at 13,400 RPM for 5 minutes to collect any undissolved powder and prevent
background.
Note: Store unused EverRed/EverBlue reagent in the dark at 4°C. Solution may be used for up to
2 months after resuspension. Centrifuge before use.
6. Prepare the SuperBoost™ EverRed/EverBlue working solution according to Table5.
IMPORTANT! Use working solution within 1 hour of preparation. Do not store excess working
solution after that time.
Table6EverRed/EverBlue working solution component volumes.
Component
Number of coverslips (18mm x 18mm)
1 5 10 50 100
EverRed/EverBlue reagent 20µL 100µL 200µL 1mL 2mL
EverRed/EverBlue development buer 80µL 400µL 800µL 4mL 8mL
7. Rinse the tissue 3 times with PBS-T.
Chapter2Methods
Perform tyramide labeling 2
Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide 15
Multiplex with primary antibodies from dierent species
After completing the first round of staining with tyramide, tissue samples can be multiplexed with
another Tyramide SuperBoost™ kit or using standard IHC/ICC protocols.
When multiplexing, use a primary antibody from a host dierent from the one used. Label the tissue
with primary antibody (mouse or rabbit host) in step5 and a color that is spectrally compatible with
the first color label. Repeat the protocol from “Perform peroxidase labeling” on page14 (include the
peroxidase quench and exclude the blocking step) as written.
Counterstain, mount, then detect the signal
1. If needed, counterstain the tissue using standard protocols. Some recommended counterstaining
reagents are listed in Table7.
Table7Products recommended for counterstain.
Counterstain target Product Cat. No.
Nucleus
Hematoxylin
MLS
Methyl Green (can discolor EverRed signal)
DAPI
Cytoplasm Eosin
2. Dehydrate with a xylene/ethanol series and mount the coverslips using an organic mountant such
as Epredia™ Cytoseal™ 60 Mountant (Cat. No. 8310-4). If aqueous mounting is preferred, use a
mountant with antifade properties such as ProLong™ Glass Antifade Mountant (Cat. No. P36982).
For optimal results, follow the instructions provided with the mountant.
3. Analyze the tissue using a compatible imaging instrument. The Tyramide SuperBoost™ system is
compatible with all types of brightfield microscopes equipped with compatible fluorescent filters.
Chapter2Methods
Multiplex with primary antibodies from dierent species
2
16 Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide
Troubleshooting
Observation Possible cause Recommended action
Excess signal Kit signal output was greater
than expected.
Optimize the primary antibody dilution.
Optimize the incubation time with the tyramide
reagent working solution.
Optimize the tyramide reagent concentration.
Low signal Kit signal output was low or
weak.
Optimize the primary antibody dilution and
incubation time.
Optimize the incubation time with the tyramide
reagent working solution.
Use antigen retrieval techniques to unmask
the signal.
Redevelop color with fresh EverRed/EverBlue
color development solution.
Low resolution or blurry signal Kit produced a poor signal
resulting in a low resolution
image.
Optimize the incubation time with the tyramide
reagent working solution.
Use antigen retrieval techniques to unmask
the signal.
High background Reaction steps were not
optimized.
Lengthen the incubation time with the H2O2
solution (step1 on page14) to decrease
endogenous peroxidase activity.
Decrease the primary antibody concentration.
Lengthen the incubation time for the blocking
step (step4 on page14).
Increase the number and/or the length of the
wash steps.
Shorten the incubation time with the tyramide
reagent working solution.
Use a lower concentration of secondary
antibody than recommended.
Dehydrate and mount using organic mountant
instead of aqueous mountant.
A
Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide 17
Signal optimization
Optimize the amount of primary antibody or probe
To optimize the amount of primary antibody or probe used in step5 on page14, we recommend testing
the following conditions:
•Slide 1: Same primary antibody or probe dilution as the standard method.
•Slide 2: Five-fold dilution of the amount used for Slide 1.
•Slide 3: Ten-fold dilution of the amount used for Slide 2 (further dilution may be necessary).
•Slide 4: Negative control (antibody or probe omitted).
Optimize the incubation time for tyramide labeling
The incubation step for the tyramide labeling reaction (step2 on page15) is crucial for getting high
resolution images with specific signal. To optimize the incubation time for this step, perform 5-, 10-, 20-,
and 30-minute incubations using positive and negative control slides.
•If nonspecific signal is present in negative controls, or if the signal is blurry in positive controls,
decrease the incubation time.
•If dim or no signal is present in positive controls, optimize the incubation time.
IMPORTANT! Longer incubation times can result in decreased signal intensity.
B
18 Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide
Ordering information
SuperBoost™ reagents available for purchase separately
Item Cat. No. Amount[1]
Tyramide SuperBoost™ EverRed HRP Substrate Kit E40963
150 slides
Tyramide SuperBoost™ EverBlue HRP Substrate Kit E40964
SuperBoost™ Goat anti-Mouse Poly HRP B40961
SuperBoost™ Goat anti-Rabbit Poly HRP B40962
Streptavidin-HRP 43-4323
[1] Amount of 150 slides is based on use of 18-mm x 18-mm coverslips.
C
Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide 19
Safety
WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user
documentation may result in personal injury or damage to the instrument or device. Ensure that
anyone using this product has received instructions in general safety practices for laboratories and
the safety information provided in this document.
·Before using an instrument or device, read and understand the safety information provided in the
user documentation provided by the manufacturer of the instrument or device.
·Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use
appropriate personal protective equipment (gloves, gowns, eye protection, and so on). To obtain
SDSs, visit thermofisher.com/support.
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel
read and practice the general safety guidelines for chemical usage, storage, and waste provided
below. Consult the relevant SDS for specific precautions and instructions:
·Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer
before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see
the "Documentation and Support" section in this document.
·Minimize contact with chemicals. Wear appropriate personal protective equipment when handling
chemicals (for example, safety glasses, gloves, or protective clothing).
·Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with
sucient ventilation (for example, fume hood).
·Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer
cleanup procedures as recommended in the SDS.
·Handle chemical wastes in a fume hood.
·Ensure use of primary and secondary waste containers. (A primary waste container holds the
immediate waste. A secondary container contains spills or leaks from the primary container.
Both containers must be compatible with the waste material and meet federal, state, and local
requirements for container storage.)
·After emptying a waste container, seal it with the cap provided.
·Characterize (by analysis if needed) the waste generated by the particular applications, reagents,
and substrates used in your laboratory.
·Ensure that the waste is stored, transferred, transported, and disposed of according to all local,
state/provincial, and/or national regulations.
·IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal
limitations may apply.
D
20 Tyramide SuperBoost™ EverRed/EverBlue HRP Kits User Guide
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Thermo Fisher Scientific Tyramide SuperBoost EverRed/EverBlue HRP Kits User guide

Brand
Thermo Fisher Scientific
Model
Tyramide SuperBoost EverRed/EverBlue HRP Kits
Type
User guide
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