HCR RNA-ISH Setup Oncore Kit Instructions
- June 1, 2024
- HCR
Table of Contents
- HCR RNA-ISH Setup Oncore Kit
- Specifications
- Product Information
- HCRTM RNA-ISH Starter Kit
- Product Usage Instructions
- FAQ
- INTRODUCTION
- HCR™ RNA-ISH Kit Information
- Overall Workflow of the HCR™ RNA-ISH Protocol
- Set Up ONCORE Pro X Protocols
- Reagent Preparation
- Overall Workflow of the HCR™ RNA-ISH + IHC/IF Protocol
- Creating an HCR™ RNA-ISH + IHC/IF Co-Detection Protocol
- Appendix
- References
- Read User Manual Online (PDF format)
- Download This Manual (PDF format)
HCR RNA-ISH Setup Oncore Kit
Specifications
- Product Name: HCRTM RNA-ISH Kit
- Compatibility: ONCORE Pro X
- Last Updated: 01/08/2024
Product Information
Upon receiving an HCRTM RNA-ISH Kit, please check all reagents and their storage conditions.
HCRTM RNA-ISH Starter Kit
| HCRTM Reagents | Amount | Storage Temperature |
|---|---|---|
| HCRTM Probe A Diluent | 1 mL | – |
| HCRTM Probe B Diluent | 1 mL | – |
Product Usage Instructions
Reagent Preparation
Follow these steps for preparing the probe solutions:
- Mix HCRTM Probe A Diluent with the provided volume of HCRTM Probe A.
- Repeat the same process for HCRTM Probe B Diluent and HCRTM Probe B.
Preparation of Probe Solutions
Estimate the required probe solutions based on the assay needs and proceed
with preparation.
Reagent Transfer and Storage
Ensure proper transfer of reagents and store them according to the
specified storage conditions.
Overall Workflow of the HCRTM RNA-ISH + IHC/IF Protocol
Follow the specific protocol outlined in the manual for combined detection
assays.
FAQ
-
How should I store the HCRTM RNA-ISH Kit reagents?
Store the reagents at the specified temperatures as indicated in the product information section of the manual. -
Can I use the HCRTM RNA-ISH Kit with other equipment?
The kit is designed for use with the ONCORE Pro X system. Compatibility with other equipment is not guaranteed.
INTRODUCTION
This Setup Guide demonstrates the use of an HCR™ RNA-ISH Kit on the ONCORE Pro X platform from Biocare Medical. Reagent preparation steps, including registering individual ONCORE Pro vials for their respective reagents, will be described in further detail. Each ONCORE Pro X run takes approximately 10 hours followed by a short post-processing of stained slides. The HCR™ RNA-ISH Kit can be used to probe and visualize RNA transcripts in FFPE tissue sections. Please read through the Setup Guide for additional information so that you can easily incorporate the HCR™ RNA-ISH assay into your current workflow.
HCR™ RNA-ISH Kit Information
Upon receiving an HCR™ RNA-ISH Kit, please check all reagents and their storage conditions.
HCR™ RNA-ISH Starter Kit
HCR™ Reagents| Amount for an HCR™ RNA-ISH Starter Kit| Storage
Temperature
---|---|---
HCR™ Probe A
PPIB/Ppib 1 – Positive Control2
| 1 mL| 2 to 8 °C
HCR™ Probe B
PPIB/Ppib 1 – Positive Control2
| 1 mL| 2 to 8 °C
HCR™ Probe A Diluent| 3 mL| 2 to 8 °C
HCR™ Probe B Diluent| 3 mL| 2 to 8 °C
HCR™ Probe A
dapB – Negative Control
| 1 mL| 2 to 8 °C
HCR™ Probe B
dapB – Negative Control
| 1 mL| 2 to 8 °C
HCR™ Probe A Diluent| 3 mL| 2 to 8 °C
HCR™ Probe B Diluent| 3 mL| 2 to 8 °C
HCR™ Membrane Stain3| 3 mL| 2 to 8 °C
HCR™ Control Slides4| 3 Slides| 2 to 8 °C
HCR™ Pretreat| 5 mL| 2 to 8 °C
HCR™ Detect A| 5 mL| 2 to 8 °C
HCR™ Detect B| 7 mL| 2 to 8 °C
HCR™ Detect C| 5 mL| 2 to 8 °C
HCR™ Detect D| 7 mL| 2 to 8 °C
HCR™ Detect E| 7 mL| 2 to 8 °C
HCR™ Detect F AP/HRP5| 7 mL| 2 to 8 °C
HCR™ Post-Process A| 5 mL| 2 to 8 °C
HCR™ Post-Process B| 7 mL| 2 to 8 °C
- Upper and lower cases are used to denote human and mouse HCR™ Probes respectively.
- These are the HCR™ Probes included in the HCR™ RNA-ISH Starter Kit, and they are provided in volumes sufficient enough to perform the assay on 10 slides each.
- The HCR™ Membrane Stain’s host species is in rabbit and is provided in a volume sufficient to perform the assay on 5 slides.
- The HCR™ Control Slides include 3 human or mouse liver FFPE tissue sections. Please allocate one slide for the positive control, one slide for the negative control, and one slide for the HCR™ RNA-ISH + IHC/IF co-detection assay using the HCR™ Membrane Stain.
- HCR™ Detect F AP is included in the HCR™ RNA-ISH AP Starter Kit, and HCR™ Detect F HRP is included in the HCR™ RNA-ISH HRP Starter Kit.
HCR™ RNA-ISH Kit
HCR™ Reagents| Amount for a 20 Slide Kit| Amount for a 90 Slide
Kit| Storage Temperature
---|---|---|---
HCR™ Probe A1| 1.75 mL| 7 mL| 2 to 8 °C
HCR™ Probe B| 1.75 mL| 7 mL| 2 to 8 °C
HCR™ Probe A Diluent| 5.25 mL| 21 mL| 2 to 8 °C
HCR™ Probe B Diluent| 5.25 mL| 21 mL| 2 to 8 °C
HCR™ Pretreat| 5 mL| 21 mL| 2 to 8 °C
HCR™ Detect A| 5 mL| 21 mL| 2 to 8 °C
HCR™ Detect B| 7 mL| 28 mL| 2 to 8 °C
HCR™ Detect C| 5 mL| 21 mL| 2 to 8 °C
HCR™ Detect D| 7 mL| 28 mL| 2 to 8 °C
HCR™ Detect E| 7 mL| 28 mL| 2 to 8 °C
HCR™ Detect F AP/HRP2| 7 mL| 28 mL| 2 to 8 °C
HCR™ Post-Process A| 5 mL| 21 mL| 2 to 8 °C
HCR™ Post-Process B3| 7 mL| 28 mL| 2 to 8 °C
- Every HCR™ Probe includes 4 components: HCR™ Probe A, HCR™ Probe B, HCR™ Probe A Diluent, and HCR™ Probe B Diluent. Please reference Page 13 for more information on how to prepare the HCR™ Probe Solution.
- HCR™ Detect F AP is included in the HCR™ RNA-ISH AP Kit, and HCR™ Detect F HRP is included in the HCR™ RNA-ISH HRP Kit.
- HCR™ Post-Process B is only needed if you are performing an HCR™ RNA-ISH + IHC/IF co-detection assay.
Required Materials for the ONCORE Pro X
The HCR™ RNA-ISH protocol requires specific materials available only from
Biocare Medical. It is essential to check the availability of these materials
prior to setting up an HCR™ RNA-ISH experiment. For more information, please
inquire with your Biocare Medical representative.
Materials from Biocare Medical
| Catalog #| Storage Temperature
ONCORE Pro 7 mL Improv Reagent Vials| ONCPR101JJ| RT
ONCORE Pro 15 mL Improv Reagent Vials|
ONCPR102JJ| RT
Dewax Solution 1, DS1 Kit| OPRI6001KT60| 2 to 8 °C
ONCORE Pro Antigen Retrieval 1 (AR1), high pH|
OPRI6006T60| 2
to 8 °C
ONCORE Pro Hematoxylin| OPRI6087T60| 2 to 8 °C
ONCORE Pro DAB Chromogen Kit1| OPRI6056KT180| 2 to 8 °C
ONCORE Pro Warp Red Chromogen Kit1| OPRI6083KT60| 2 to 8 °C
ONCORE Pro Rabbit HRP Detection2| OPRI6008T60| 2 to 8 °C
ONCORE Pro Rabbit AP Detection2| OPRI6043T60| 2 to 8 °C
ONCORE Pro Mouse HRP Detection2| OPRI6007T60| 2 to 8 °C
ONCORE Pro Mouse AP Detection2| OPRI6044T60| 2 to 8 °C
- The HCR™ RNA-ISH protocol uses DAB and Warp Red chromogens for HRP and AP detection, respectively.
- The Rabbit or Mouse Polymer Detection Kits are only needed for ISH + IHC/IF co-detection assays.
User-Supplied Materials
Materials from Other Vendors
| Supplier| Comment
FFPE Sample Slides| Any| SuperFrost or SuperFrost® Plus slides are recommended
for best results
Propar (xylene substitute)| Fisher Scientific| Xylene may be substituted
Drying Oven| Any| Capable of maintaining temperature at ~60 °C
BioCare EcoMount| Biocare Medical| Mounting medium compatible with all Biocare
chromogens
Cytoseal| Any| Suitable mounting medium for HRP-driven chromogens
ProLong™ Gold Antifade Mountant with DAPI| Thermo Fisher| Mounting medium
compatible with RNA-FISH and IF staining
Cover Glass| Any| Dimension depends on the size of the tissue
100% Ethanol| Any| None
Tyramide Dyes| Any| See Appendix E for Recommendations
Overall Workflow of the HCR™ RNA-ISH Protocol
As mentioned earlier, each ONCORE Pro X run takes approximately 10 hours. The timeline above only accounts for 7 hours and 27 minutes of this run (depending on whether you’re performing the assay for chromogenic or fluorescent detection), as the remaining time comes from the additional ONCORE Pro X washing steps.
Set Up ONCORE Pro X Protocols
The four main steps in setting up an ONCORE Pro X protocol for an HCR™ RNA-ISH run are:
- Register HCR™ RNA-ISH reagents in the ONCORE Pro X software
- Create an HCR™ RNA-ISH protocol using an existing IHC staining protocol as a template
- Select protocols for individual slide modules
- Scan and map reagents and initiate the staining process
Register HCR™ RNA-ISH Reagents in the ONCORE Pro X Software
In order to create ONCORE Pro X staining protocols, reagent names associated
with the HCR™ Reagents need to be registered in the ONCORE Pro X System
Software. The table below lists all the reagents that need to be registered
for the HCR™ RNA-ISH assay.
Oncore Pro Reagent Vials
Name| Type of Container 1| Reagent Type| Viscosity Level
HCR™ Pretreat| 7 mL or 15 mL ONCORE Pro Reagent Vial| Special| 1
HCR™ Probe A| 7 mL or 15 mL ONCORE Pro Reagent Vial| CISH RNA| 4
HCR™ Probe B| 7 mL or 15 mL ONCORE Pro Reagent Vial| CISH RNA| 4
HCR™ Detect A| 7 mL or 15 mL ONCORE Pro Reagent Vial| Special| 1
HCR™ Detect B| 7 mL or 15 mL ONCORE Pro Reagent Vial| Special| 1
HCR™ Detect C| 7 mL or 15 mL ONCORE Pro Reagent Vial| Special| 1
HCR™ Detect D| 7 mL or 15 mL ONCORE Pro Reagent Vial| Special| 1
HCR™ Detect E| 7 mL or 15 mL ONCORE Pro Reagent Vial| Special| 1
HCR™ Detect F HRP/AP| 7 mL or 15 mL ONCORE Pro Reagent Vial| Special| 1
HCR™ Post-Process A| 7 mL or 15 mL ONCORE Pro Reagent Vial| Special| 1
HCR™ Post-Process B2| 7 mL or 15 mL ONCORE Pro Reagent Vial| Special| 1
Primary Antibody2| 7 mL ONCORE Pro Reagent Vial| Special| 1
We recommend using 7 mL and 15 mL Oncore Pro Reagent Vials for 20-slide and
90-slide kits, respectively.
These reagents are only required for running an HCR™ RNA-ISH + IHC/IF co-
detection assay.
- To register HCR™ RNA-ISH reagents, open the ONCORE Pro X System Software and go to: System utilities → Reagent Editor → Select Reagent Type (e.g., CISH RNA) → Add New → Enter the HCR™ Reagent names → Check/change the viscosity level → Save
- Repeat Step 1 for all other HCR™ Reagents listed in the table above.
Create an HCR™ RNA-ISH Protocol Using an Existing IHC Staining Protocol as a Template
-
The Advanced Protocol Editor is a software that enables you to create custom protocols with the help of a protocol template. After opening the Advanced Protocol Editor, select one of the pre-existing IHC protocols such as Ms HRP Temp1 and use it as a template.
Note: This template already includes the dewax and target retrieval steps used in the HCR™ RNA-ISH protocols. The target retrieval temperature should be set at 95 C. -
There are three single-plex RNA-ISH protocols:
- HCR™ RNA-CISH HRP (see pages 17-19)
- HCR™ RNA-CISH AP (see pages 17-19)
- HCR™ RNA-FISH (see pages 20-22)
| HCR™ RNA-CISH HRP| HCR™ RNA-CISH AP| HCR™ RNA-FISH
---|---|---|---
Main Differences:
|
Uses HCR™ Detect F HRP and an HRP-driven chromogen (e.g., DAB).
|
Uses HCR™ Detect F AP and an AP-driven chromogen (e.g., Warp Red).
|
Uses third-party Tyramide dyes. Hematoxylin and HCR™ Post-Process A should be omitted.
Note: Please see the Appendix for detailed staining protocols for each assay type (Appendices A and B) and third-party recommended Tyramide dyes (Appendix E).
- Slides could be labeled by either (1) preparing label options (see the ONCORE User Manual for more information) or (2) handwriting directly on the slides.
- Click on Slide Modules. This is where the slides will be stained after assigning protocols by clicking on Assign Protocols. Staining protocols created from the Advanced Protocol Editor will show up from the drop-down menu. Select the appropriate protocols (HCR™ RNA-CISH HRP is selected in the following figure).
Scan and Map Reagents and Initiate the Staining Process
Before initiating a run, all reagents used by the previously programmed
protocols need to be either (1) scanned or (2) manually mapped. We recommend
placing ready-to-use (RTU) Biocare reagents in the first two rows (i.e.,
positions 1-10), and the rest of the HCR™ Reagents in the other rows (i.e.,
positions 11-39).
- Click on Scan Reagents to allow the instrument to scan all the Biocare reagents. Once all the Biocare reagents have been scanned, click on Stop to conclude the scanning process. The unscanned HCR™ Reagents will appear in red.
- Map the HCR™ reagents to the proper positions by clicking on the Reagent Name First in the table. Then, click reagent position in the left panel. Following the manual assignment, the outcome should resemble the image provided below.
- Initiate the HCR™ RNA-ISH run immediately by selecting Start Staining Process. Alternatively, Delay Start Staining Process allows you to schedule the run to finish at a specific time.
Post-Processing for HRP-Based Detection
After slides are unloaded from the ONCORE Pro X, we recommend washing the
slides thoroughly with tap water. Dehydrate by immersing the slides in 95%
ethanol for 3 minutes twice followed by 100% ethanol for 3 minutes twice.
Then, immerse the slides in a xylene (or xylene substitute) solution for 5
minutes and mount one slide at a time with Cytoseal (or any other xylene-based
mounting medium) or EcoMount. Allow slides to air dry for 5 minutes before
imaging.
Post-Processing for AP-Based Detection
After slides are unloaded from the ONCORE Pro X, we recommend washing the
slides thoroughly with tap water. Bake the slides for at least 15 minutes (or
until dry) at 60 °C. Dip the slide in xylene (or xylene substitute) for 1-3
seconds and immediately apply EcoMount for cover-slipping.
Post-Processing for Fluorescent Detection
After slides are unloaded from the ONCORE Pro X, we recommend immersing the
slides in 1 x PBST/TBST for 3 minutes. Mount one slide at a time with the
ProLong™ Gold Antifade mounting medium (or any other suitable mounting medium
that you are familiar with).
Reagent Preparation
The three main steps to prepare for an HCR™ RNA-ISH run are:
- Prepare Probe Solutions
- Estimate the amount of probe solutions needed (this is an optional step)
- Transfer all HCR™ Reagents to the ONCORE Vials (7 mL or 15 mL)
Preparation of Probe Solutions
HCR™ Probe Hybridization is a two-step process that requires two separate
probe solutions. HCR™ Probe A Solution and HCR™ Probe B Solution can be
prepared separately by mixing HCR™ Probe A with HCR™ Probe A Diluent and HCR™
Probe B with HCR™ Probe B Diluent, respectively. We recommend transferring the
entirety of HCR™ Probe A and HCR™ Probe B into the HCR™ Probe A Diluent and
HCR™ Probe B Diluent bottles. After the solutions have been transferred, you
can either vortex the bottles or invert the bottle 4-5 times to ensure proper
mixture.
Estimation of Required Probe Solutions
- To calculate the exact quantity of probe solution needed, it is important to consider the dead volume of an ONCORE Vial. The volumes needed for N number of slides can be calculated using the formula for the ONCORE Pro X listed below:
- 2201.08N + 500 µL (dead volume) for a 7 mL ONCORE Vial
- 2201.08N + 1000 µL (dead volume) for a 15 mL ONCORE Vial
- The amount of each probe solution needed per slide is 220 µL. The table below shows the volume of probe solution needed for staining 3 slides while taking the dead volumes into account.
Container Type| Probe Solution Volume| Amount of HCR™ Reagent
---|---|---
7 mL ONCORE Vial
|
1213 µL
(2201.08 3 + 500)
| Transfer 1213 µL of probe solution to the registered probe vials (e.g., PPIB HCR™ Probe A and PPIB HCR™ Probe B)
15 mL ONCORE Vial
|
1713 µL
(2201.08 3 + 1000)
| Transfer 1713 µL of probe solution to the registered probe vials (e.g., PPIB HCR™ Probe A and PPIB HCR™ Probe B)
NOTE: You can choose to transfer all probe solutions (~7 mL for each probe) to the respective ONCORE Pro Vials. Store the Vials at 4 °C after each run.
Reagent Transfer and Storage
Instructions for reagent transfer can be found in the table below. Please
transfer all HCR™ RNA-ISH reagents to their name-associated vials. Store all
ONCORE Pro X Vials in 4C fridge after use.
| Oncore Vials | Instructions 1 | Storage Conditions |
|---|---|---|
| HCR™ Pretreat | Transfer the entirety of HCR™ Pretreat to the 7 mL or 15 mL | |
| Vial | Store at 4 °C after use |
HCR™ Probe A Solution
| Transfer the entirety of or the calculated amount of HCR™ Probe A Solution to the 7 mL or 15 mL Vial|
Store at 4 °C after use
HCR™ Probe B Solution
| Transfer the entirety of or the calculated amount of HCR™ Probe B Solution to the 7 mL or 15 mL Vial|
Store at 4 °C after use
HCR™ Detect A| Transfer entirety of HCR™ Detect A to the 7 mL or 15 mL Vial|
Store at 4 °C after use
HCR™ Detect B| Transfer entirety of HCR™ Detect B to the 7 mL or 15 mL Vial|
Store at 4 °C after use
HCR™ Detect C| Transfer entirety of HCR™ Detect C to the 7 mL or 15 mL Vial|
Store at 4 °C after use
HCR™ Detect D| Transfer entirety of HCR™ Detect D to the 7 mL or 15 mL Vial|
Store at 4 °C after use
HCR™ Detect E| Transfer entirety of HCR™ Detect E to the 7 mL or 15 mL Vial|
Store at 4 °C after use
HCR™ Detect F AP| Transfer entirety of HCR™ Detect F AP to the 7 mL or 15 mL
Vial| Store at 4 °C after use
HCR™ Detect F HRP| Transfer entirety of HCR™ Detect F HRP to the 7 mL or 15 mL
Vial| Store at 4 °C after use
HCR™ Post-Process A| Transfer entirety of HCR™ Post-Process A to the 7 mL or
15 mL Vial| Store at 4 °C after use
HCR™ Post-Process B| Transfer entirety of HCR™ Post-Process B to the 7 mL or
15 mL Vial| Store at 4 °C after use
Primary Antibody| Transfer entirety of HCR™ Membrane Stain2 to the 7 mL Vial|
Store at 4 °C after use
- Note: Use 7 mL and 15 mL vials for 20-slide and 90-slide kits respectively. The HCR™ Reagent volumes for 90-slide kits will be more than 15 mL. You can refill the 15 mL vials as needed after each run.
- The HCR™ Membrane Stain is only provided in the HCR™ RNA-ISH Starter Kit.
Other important notes:
- Please avoid excessive bubbles when transferring HCR™ Probe and HCR™ Detect Solutions to the 7 mL or 15 mL ONCORE Vials.
- If the reagent-filled ONCORE Vials have not been used for more than a week, it is highly recommended to gently invert the vials several times with the lid closed to ensure solution uniformity before next use.
- Please note that the use of Warp Red and Fast Red Biocare chromogens can sometimes result in non-uniform staining for tissues placed close to the injection port. To mitigate the risk of non-uniform staining, Biocare suggests placing your tissue samples in a designated area, as illustrated in the image below.
Overall Workflow of the HCR™ RNA-ISH + IHC/IF Protocol
Creating an HCR™ RNA-ISH + IHC/IF Co-Detection Protocol
The additional reagents needed to run an HCR™ RNA-ISH + IHC/IF co-detection assay are:
- HCR™ Post-Process B
- User-provided primary antibody (or the HCR™ Membrane Stain from the HCR™ RNA-ISH Starter Kit)
- Anti-Ms, Rb AP-, or HRP-conjugated polymer detection kits from Biocare Medical
- HRP- or AP-driven chromogens for IHC or third-party Tyramide-conjugated fluorophores for IF
Create an HCR™ RNA-CISH + IHC Co-Detection Protocol
This co-detection protocol can be created using the previously programmed
HCR™ RNA-CISH HRP/AP protocol as a template. Please follow the detailed HCR™
RNA-CISH + IHC co-detection protocol (see pages 23 to 26) in Appendix C for
additional steps.
Note: HCR™ Post-Process B (steps 33 to 34) is not needed if HCR™ RNA-CISH
HRP detection is followed by AP-driven IHC detection.
Create an HCR™ RNA-FISH + IF Co-Detection Protocol
This co-detection protocol can be created using the previously programmed HCR™
RNA-FISH protocol as a template. Please follow the detailed HCR™ RNA-FISH + IF
co-detection protocol (see pages 27-29) in Appendix D for additional steps.
Note: You will need to provide another Tyramide dye for IF that is
spectrally different from the Tyramide dye used for HCR™ RNA-FISH. See
Appendix E for a list of recommended third-party Tyramide dyes and their
suggested starting concentrations.
Appendix
Appendix A: Detailed Staining Protocol for HCR™ RNA-CISH [Chromogenic Detection]
Step No.| Reagent Type| Reagent name| Reagent Vol. (µL)| Total Inc. Time| Temp (°C)| Heat Inc. Time| __
Agitation
| __
#
| Wash Buffer Vol.
(µL)
---|---|---|---|---|---|---|---|---|---
__
1
| __
Dewax
| __
DS1 (No
extraction)
| __
240
| __
00:05:00
| __
65.00
| __
00:04:30
| Lid up: 1 sec (injection) Lid down: 59
sec (A4)
| __
0
| __
85
__
2
| __
Dewax
| __
DS2-50
| __
200
| __
00:04:30
| __
58.00
| __
00:02:30
| Lid up: 1 sec (injection) Lid down: 59
sec (A4)
| __
1
| __
220
__
3
| __
Retrieval
| __
AR1, high pH
| __
370
| __
00:32:00
| __
95.00
| __
00:27:00
| Lid up: 15 min (A3)
Lid down: 17
min (A4)
| __
1
| __
200
__
4
| __
Buffer
| __
System Fluid
| __
220
| __
00:04:20
| __
33.00
| __
00:59:00
| Lid up: 1 sec (injection) Lid down: 59
sec (A4)
| __
1
| __
85
__
5
| __
Buffer
| __
System Fluid
| __
220
| __
00:00:10
| __
RT
| __
00:00:00
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
220
__
6
| __
Special
| __
HCR™
Pretreat
| __
220
| __
00:05:00
| __
RT
| __
00:05:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
1
| __
240
__
7
| __
Buffer
| __
System Fluid
| __
220
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
200
__
8
| __
CISH RNA
| HCR™ Probe A| __
220
| __
01:30:00
| __
45.00
| __
01:30:00
| Lid up: 1 sec (injection) Lid down: 30
min (A4)
| __
1
| __
240
__
9
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
10
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
11
| __
Buffer
| __
System Fluid
| __
240
| __
00:05:00
| __
45.00
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 85
sec (A4)
| __
0
| __
85
__
12
| __
CISH RNA
| HCR™ Probe B| __
220
| __
01:30:00
| __
45.00
| __
01:30:00
| Lid up: 1 sec (injection) Lid down: 30
min (A4)
| __
1
| __
240
__
13
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
14
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
---|---|---|---|---|---|---|---|---|---
__
15
| __
Buffer
| __
System Fluid
| __
240
| __
00:05:00
| __
45.00
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 85
sec (A4)
| __
0
| __
85
__
16
| __
Special
| HCR™ Detect A| __
220
| __
00:03:00
| __
42.00
| __
00:03:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
0
| __
240
__
17
| __
Special
| HCR™ Detect B| __
220
| __
01:30:00
| __
42.00
| __
01:30:00
| Lid up: 1 sec (injection) Lid down: 30
min (A4)
| __
1
| __
240
__
18
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
42.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
19
| __
Buffer
| __
System Fluid
| __
240
| __
00:05:00
| __
42.00
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
1
| __
240
__
20
| __
Special
| HCR™ Detect C| __
220
| __
00:05:00
| __
RT
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
0
| __
85
__
21
| __
Special
| HCR™ Detect D| __
220
| __
00:20:00
| __
RT
| __
00:20:00
| Lid up: 1 sec (injection)
Lid down: 3 min
(A4)
| __
1
| __
240
__
22
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 15
sec (A4)
| __
0
| __
85
__
23
| __
Buffer
| __
System Fluid
| __
240
| __
00:03:00
| __
RT
| __
00:03:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
0
| __
85
__
24
| __
Special
| HCR™ Detect E| __
220
| __
00:12:00
| __
RT
| __
00:12:00
| Lid up: 1 sec (injection)
Lid down: 3 min
(A4)
| __
1
| __
240
__
25
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
26
| __
Buffer
| __
System Fluid
| __
240
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
85
__
27*
| __
Special
| __
HCR™ Detect F HRP*
| __
220
| __
00:20:00
| __
RT
| __
00:20:00
| Lid up: 1 sec (injection)
Lid down: 3 min
(A4)
| __
1
| __
240
__
28
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
29
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
---|---|---|---|---|---|---|---|---|---
__
30*
| __
Detection
| __
DAB*
| __
220
| __
00:08:40
| __
37.00
| __
00:08:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
1
| __
240
__
31
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 15
sec (A4)
| __
0
|
__
32
| __
Others
| __
Hematoxylin
| __
220
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 30
sec (A4)
| __
2
| __
240
__
33
| __
Buffer
| __
System Fluid
| __
220
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 15
sec (A4)
| __
0
| __
85
__
34
| __
Special
| __
HCR™ Post- Process A
| __
220
| __
00:03:00
| __
RT
| __
00:03:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
1
| __
240
__
35
| __
Buffer
| __
System Fluid
| __
130
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 15
sec (Down)
| __
0
| __
85
- For AP-based detection, please make changes to steps 27 and 30 as follows:
__
27*
| __
Special
| __
HCR™ Detect F AP*
| __
220
| __
00:20:00
| __
RT
| __
00:20:00
| Lid up: 1 sec (injection)
Lid down: 3 min
(A4)
| __
1
| __
240
---|---|---|---|---|---|---|---|---|---
__
30*
| __
Detection
| __
Warp Red*
| __
220
| __
00:07:00
| __
RT
| __
00:09:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
1
| __
240
Appendix B: Detailed Staining Protocol for HCR™ RNA-FISH [Fluorescent Detection]
Step No.| Reagent Type| Reagent name| Reagent Vol. (µL)| Total Inc. Time| Temp (°C)| Heat Inc. Time| __
Agitation
| __
#
| Wash Buffer Vol.
(µL)
---|---|---|---|---|---|---|---|---|---
__
1
| __
Dewax
| __
DS1 (No
extraction)
| __
240
| __
00:05:00
| __
65.00
| __
00:04:30
| Lid up: 1 sec (injection) Lid down: 59
sec (A4)
| __
0
| __
85
__
2
| __
Dewax
| __
DS2-50
| __
200
| __
00:04:30
| __
58.00
| __
00:02:30
| Lid up: 1 sec (injection) Lid down: 59
sec (A4)
| __
1
| __
220
__
3
| __
Retrieval
| __
AR1, high pH
| __
370
| __
00:32:00
| __
95.00
| __
00:27:00
| Lid up: 15 min (A3)
Lid down: 17
min (A4)
| __
1
| __
200
__
4
| __
Buffer
| __
System Fluid
| __
220
| __
00:04:20
| __
33.00
| __
00:59:00
| Lid up: 1 sec (injection) Lid down: 59
sec (A4)
| __
1
| __
85
__
5
| __
Buffer
| __
System Fluid
| __
220
| __
00:00:10
| __
RT
| __
00:00:00
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
220
__
6
| __
Special
| __
HCR™
Pretreat
| __
220
| __
00:05:00
| __
RT
| __
00:05:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
1
| __
240
__
7
| __
Buffer
| __
System Fluid
| __
220
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
200
__
8
| __
CISH RNA*
| HCR™ Probe A| __
220
| __
01:30:00
| __
45.00
| __
01:30:00
| Lid up: 1 sec (injection) Lid down: 30
min (A4)
| __
1
| __
240
__
9
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
10
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
11
| __
Buffer
| __
System Fluid
| __
240
| __
00:05:00
| __
45.00
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 85
sec (A4)
| __
0
| __
85
__
12
| __
CISH RNA*
| HCR™ Probe B| __
220
| __
01:30:00
| __
45.00
| __
01:30:00
| Lid up: 1 sec (injection) Lid down: 30
min (A4)
| __
1
| __
240
__
13
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
14
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
15
| __
Buffer
| __
System Fluid
| __
240
| __
00:05:00
| __
45.00
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 85
sec (A4)
| __
0
| __
85
---|---|---|---|---|---|---|---|---|---
__
16
| __
Special
| __
HCR™ Detect A
| __
220
| __
00:03:00
| __
42.00
| __
00:03:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
0
| __
240
__
17
| __
Special
| HCR™ Detect B| __
220
| __
01:30:00
| __
42.00
| __
01:30:00
| Lid up: 1 sec (injection) Lid down: 30
min (A4)
| __
1
| __
240
__
18
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
42.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
19
| __
Buffer
| __
System Fluid
| __
240
| __
00:05:00
| __
42.00
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
1
| __
240
__
20
| __
Special
| __
HCR™ Detect C
| __
220
| __
00:05:00
| __
RT
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
0
| __
85
__
21
| __
Special
| HCR™ Detect D| __
220
| __
00:20:00
| __
RT
| __
00:20:00
| Lid up: 1 sec (injection)
Lid down: 3 min
(A4)
| __
1
| __
240
__
22
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 15
sec (A4)
| __
0
| __
85
__
23
| __
Buffer
| __
System Fluid
| __
240
| __
00:03:00
| __
RT
| __
00:03:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
0
| __
85
__
24
| __
Special
| __
HCR™ Detect E
| __
220
| __
00:12:00
| __
RT
| __
00:12:00
| Lid up: 1 sec (injection)
Lid down: 3 min
(A4)
| __
1
| __
240
__
25
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
26
| __
Buffer
| __
System Fluid
| __
240
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
85
__
27
| __
Special
| __
HCR™ Detect F HRP
| __
220
| __
00:20:00
| __
RT
| __
00:20:00
| Lid up: 1 sec (injection)
Lid down: 3 min
(A4)
| __
1
| __
240
__
28
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
29
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
30*
| __
Special
| __
Tyramide Dye 1**
| __
220
| __
00:15:00#
| __
RT
| __
00:17:00
| Lid up: 1 sec (injection)
Lid down: 2 min
30 sec (A4)
| __
2
| __
240
---|---|---|---|---|---|---|---|---|---
__
31
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:02:00
| Lid up: 1 sec (injection) Lid down: 15
sec (A4)
| __
0
| __
240
__
32
| __
Buffer
| __
System Fluid
| __
130
| __
00:01:00
| __
RT
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 15
sec (Down)
| __
0
| __
85
- Please keep in mind that while CISH RNA is used here as the reagent type, the HCR™ Probes will still work as expected for fluorescent detection.
- The incubation time of the Tyramide dye listed here is only used for reference and you will need to optimize the actual incubation time
Appendix C: Detailed Staining Protocol for HCR™ RNA-CISH + IHC Co- Detection
Step No.| Reagent Type| Reagent name| Reagent Vol. (µL)| Total Inc. Time| Temp (°C)| Heat Inc. Time| __
Agitation
| __
#
| Wash Buffer Vol.
(µL)
---|---|---|---|---|---|---|---|---|---
__
1
| __
Dewax
| __
DS1 (No
extraction)
| __
240
| __
00:05:00
| __
65.00
| __
00:04:30
| Lid up: 1 sec (injection) Lid down: 59
sec (A4)
| __
0
| __
85
__
2
| __
Dewax
| __
DS2-50
| __
200
| __
00:04:30
| __
58.00
| __
00:02:30
| Lid up: 1 sec (injection) Lid down: 59
sec (A4)
| __
1
| __
220
__
3
| __
Retrieval
| __
AR1, high pH
| __
370
| __
00:32:00
| __
95.00
| __
00:27:00
| Lid up: 15 min (A3)
Lid down: 17
min (A4)
| __
1
| __
200
__
4
| __
Buffer
| __
System Fluid
| __
220
| __
00:04:20
| __
33.00
| __
00:59:00
| Lid up: 1 sec (injection) Lid down: 59
sec (A4)
| __
1
| __
85
__
5
| __
Buffer
| __
System Fluid
| __
220
| __
00:00:10
| __
RT
| __
00:00:00
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
220
__
6
| __
Special
| __
HCR™
Pretreat
| __
220
| __
00:05:00
| __
RT
| __
00:05:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
1
| __
240
__
7
| __
Buffer
| __
System Fluid
| __
220
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
200
__
8
| __
CISH RNA
| HCR™ Probe A| __
220
| __
01:30:00
| __
45.00
| __
01:30:00
| Lid up: 1 sec (injection) Lid down: 30
min (A4)
| __
1
| __
240
__
9
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
10
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
11
| __
Buffer
| __
System Fluid
| __
240
| __
00:05:00
| __
45.00
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 85
sec (A4)
| __
0
| __
85
__
12
| __
CISH RNA
| HCR™ Probe B| __
220
| __
01:30:00
| __
45.00
| __
01:30:00
| Lid up: 1 sec (injection) Lid down: 30
min (A4)
| __
1
| __
240
__
13
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
14
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
15
| __
Buffer
| __
System Fluid
| __
240
| __
00:05:00
| __
45.00
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 85
sec (A4)
| __
0
| __
85
---|---|---|---|---|---|---|---|---|---
__
16
| __
Special
| __
HCR™ Detect A
| __
220
| __
00:03:00
| __
42.00
| __
00:03:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
0
| __
240
__
17
| __
Special
| HCR™ Detect B| __
220
| __
01:30:00
| __
42.00
| __
01:30:00
| Lid up: 1 sec (injection) Lid down: 30
min (A4)
| __
1
| __
240
__
18
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
42.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
19
| __
Buffer
| __
System Fluid
| __
240
| __
00:05:00
| __
42.00
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
1
| __
240
__
20
| __
Special
| __
HCR™ Detect C
| __
220
| __
00:05:00
| __
RT
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
0
| __
85
__
21
| __
Special
| HCR™ Detect D| __
220
| __
00:20:00
| __
RT
| __
00:20:00
| Lid up: 1 sec (injection)
Lid down: 3 min
(A4)
| __
1
| __
240
__
22
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 15
sec (A4)
| __
0
| __
85
__
23
| __
Buffer
| __
System Fluid
| __
240
| __
00:03:00
| __
RT
| __
00:03:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
0
| __
85
__
24
| __
Special
| __
HCR™ Detect E
| __
220
| __
00:12:00
| __
RT
| __
00:12:00
| Lid up: 1 sec (injection)
Lid down: 3 min
(A4)
| __
1
| __
240
__
25
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
26
| __
Buffer
| __
System Fluid
| __
240
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
85
__
27
| __
Special
| __
HCR™ Detect F AP
| __
220
| __
00:20:00
| __
RT
| __
00:20:00
| Lid up: 1 sec (injection)
Lid down: 3 min
(A4)
| __
1
| __
240
__
28
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
29
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
30
| __
Detection
| __
Warp Red
| __
220
| __
00:07:00
| __
RT
| __
00:17:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
2
| __
240
---|---|---|---|---|---|---|---|---|---
__
31
| __
Buffer
| __
System Fluid
| __
130
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A7)
| __
0
| __
85
__
32
| __
Buffer
| __
System Fluid
| __
240
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
85
__
33
| __
Special
| HCR™ Post- Process B| __
220
| __
00:15:00
| __
42.00
| __
00:15:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
1
| __
240
__
34
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
35
| __
Special
| __
Primary Antibody
| __
130
| __
00:30:00
| __
RT
| __
00:30:00
| Lid up: 1 sec (injection)
Lid down: 2 min
30 sec (A7)
| __
1
| __
240
__
36
| __
Buffer
| __
System Fluid
| __
240
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
85
__
37
| __
Detection
| __
Rabbit HRP*
| __
130
| __
00:20:00
| __
RT
| __
00:20:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A7)
| __
1
| __
240
__
38
| __
Buffer
| __
System Fluid
| __
240
| __
00:04:20
| __
35
| __
00:04:00
| Lid up: 1 sec (injection)
Lid down: 2 min
(A4)
| __
0
| __
85
__
39
| __
Buffer
| __
System Fluid
| __
240
| __
00:04:20
| __
35
| __
00:04:00
| Lid up: 1 sec (injection)
Lid down: 2 min
(A4)
| __
1
| __
240
__
40
| __
Detection
| __
DAB**
| __
220
| __
00:08:40
| __
37.00
| __
00:08:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
1
| __
240
__
41
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 15
sec (A4)
| __
0
|
__
42
| __
Others
| __
Hematoxylin
| __
220
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 30
sec (A4)
| __
2
| __
240
__
43
| __
Buffer
| __
System Fluid
| __
220
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 15
sec (A4)
| __
0
| __
85
__
44
| __
Special
| HCR™ Post- Process A| __
220
| __
00:03:00
| __
RT
| __
00:03:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
1
| __
240
__
45
| __
Buffer
| __
System Fluid
| __
130
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 15
sec (Down)
| __
0
| __
85
---|---|---|---|---|---|---|---|---|---
- Choose the appropriate ONCORE Polymer Detection based on the species (Rabbit or Mouse) of the primary antibody and the type of chromogen (HRP- or AP-driven) used.
- DAB is used here as an example. You may use other chromogens instead.
Appendix D: Detailed Staining Protocol for HCR™ RNA-FISH + IF Co- Detection
Step No.| Reagent Type| Reagent name| Reagent Vol. (µL)| Total Inc. Time| Temp (°C)| Heat Inc. Time| __
Agitation
| __
#
| Wash Buffer Vol.
(µL)
---|---|---|---|---|---|---|---|---|---
__
1
| __
Dewax
| __
DS1 (No
extraction)
| __
240
| __
00:05:00
| __
65.00
| __
00:04:30
| Lid up: 1 sec (injection) Lid down: 59
sec (A4)
| __
0
| __
85
__
2
| __
Dewax
| __
DS2-50
| __
200
| __
00:04:30
| __
58.00
| __
00:02:30
| Lid up: 1 sec (injection) Lid down: 59
sec (A4)
| __
1
| __
220
__
3
| __
Retrieval
| __
AR1, high pH
| __
370
| __
00:32:00
| __
95.00
| __
00:27:00
| Lid up: 15 min (A3)
Lid down: 17
min (A4)
| __
1
| __
200
__
4
| __
Buffer
| __
System Fluid
| __
220
| __
00:04:20
| __
33.00
| __
00:59:00
| Lid up: 1 sec (injection) Lid down: 59
sec (A4)
| __
1
| __
85
__
5
| __
Buffer
| __
System Fluid
| __
220
| __
00:00:10
| __
RT
| __
00:00:00
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
220
__
6
| __
Special
| __
HCR Pretreat
| __
220
| __
00:05:00
| __
RT
| __
00:05:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
1
| __
240
__
7
| __
Buffer
| __
System Fluid
| __
220
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
200
__
8
| __
CISH RNA*
| HCR™ Probe A| __
220
| __
01:30:00
| __
45.00
| __
01:30:00
| Lid up: 1 sec (injection) Lid down: 30
min (A4)
| __
1
| __
240
__
9
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
10
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
11
| __
Buffer
| __
System Fluid
| __
240
| __
00:05:00
| __
45.00
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 85
sec (A4)
| __
0
| __
85
__
12
| __
CISH RNA*
| HCR™ Probe B| __
220
| __
01:30:00
| __
45.00
| __
01:30:00
| Lid up: 1 sec (injection) Lid down: 30
min (A4)
| __
1
| __
240
__
13
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
14
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
45.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
15
| __
Buffer
| __
System Fluid
| __
240
| __
00:05:00
| __
45.00
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 85
sec (A4)
| __
0
| __
85
---|---|---|---|---|---|---|---|---|---
__
16
| __
Special
| __
HCR™ Detect A
| __
220
| __
00:03:00
| __
42.00
| __
00:03:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
0
| __
240
__
17
| __
Special
| HCR™ Detect B| __
220
| __
01:30:00
| __
42.00
| __
01:30:00
| Lid up: 1 sec (injection) Lid down: 30
min (A4)
| __
1
| __
240
__
18
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
42.00
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
19
| __
Buffer
| __
System Fluid
| __
240
| __
00:05:00
| __
42.00
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
1
| __
240
__
20
| __
Special
| __
HCR™ Detect C
| __
220
| __
00:05:00
| __
RT
| __
00:05:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
0
| __
85
__
21
| __
Special
| HCR™ Detect D| __
220
| __
00:20:00
| __
RT
| __
00:20:00
| Lid up: 1 sec (injection)
Lid down: 3 min
(A4)
| __
1
| __
240
__
22
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 15
sec (A4)
| __
0
| __
85
__
23
| __
Buffer
| __
System Fluid
| __
240
| __
00:03:00
| __
RT
| __
00:03:00
| Lid up: 1 sec (injection)
Lid down: 1 min
(A4)
| __
0
| __
85
__
24
| __
Special
| __
HCR™ Detect E
| __
220
| __
00:12:00
| __
RT
| __
00:12:00
| Lid up: 1 sec (injection)
Lid down: 3 min
(A4)
| __
1
| __
240
__
25
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
26
| __
Buffer
| __
System Fluid
| __
240
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
85
__
27
| __
Special
| __
HCR™ Detect F HRP
| __
220
| __
00:20:00
| __
RT
| __
00:20:00
| Lid up: 1 sec (injection)
Lid down: 3 min
(A4)
| __
1
| __
240
__
28
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
29
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
30
| __
Special
| __
Tyramide Dye 1
| __
220
| __
00:15:00
| __
RT
| __
00:17:00
| Lid up: 1 sec (injection)
Lid down: 2 min
30 sec (A4)
| __
2
| __
240
---|---|---|---|---|---|---|---|---|---
__
31
| __
Buffer
| __
System Fluid
| __
130
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A7)
| __
0
| __
85
__
32
| __
Buffer
| __
System Fluid
| __
240
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
85
__
33
| __
Special
| HCR™ Post- Process B| __
220
| __
00:15:00
| __
42.00
| __
00:15:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A4)
| __
1
| __
240
__
34
| __
Buffer
| __
System Fluid
| __
240
| __
00:01:00
| __
RT
| __
00:01:00
| Lid up: 1 sec (injection) Lid down: 25
sec (A4)
| __
0
| __
85
__
35
| __
Special
| __
Primary Antibody
| __
130
| __
00:30:00
| __
RT
| __
00:30:00
| Lid up: 1 sec (injection)
Lid down: 2 min
30 sec (A7)
| __
1
| __
240
__
36
| __
Buffer
| __
System Fluid
| __
240
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
85
__
37
| __
Detection
| __
Rabbit HRP
| __
130
| __
00:20:00
| __
RT
| __
00:20:00
| Lid up: 1 sec (injection) Lid down: 90
sec (A7)
| __
1
| __
240
__
38
| __
Buffer
| __
System Fluid
| __
240
| __
00:04:20
| __
35
| __
00:04:00
| Lid up: 1 sec (injection)
Lid down: 2 min
(A4)
| __
0
| __
85
__
39
| __
Buffer
| __
System Fluid
| __
240
| __
00:04:20
| __
35
| __
00:04:00
| Lid up: 1 sec (injection)
Lid down: 2 min
(A4)
| __
1
| __
240
__
40
| __
Special
| Tyramide Dye 2| __
220
| __
00:15:00
| __
RT
| __
00:17:00
| Lid up: 1 sec (injection)
Lid down: 2 min
30 sec (A4)
| __
2
| __
240
__
41
| __
Buffer
| __
System Fluid
| __
240
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(A4)
| __
0
| __
85
__
42
| __
Buffer
| __
System Fluid
| __
130
| __
00:00:10
| __
RT
| __
00:00:10
| Lid up: 1 sec (injection)
Lid down: 1 sec
(Down)
| __
0
| __
85
Appendix E: Third-Party Recommended Tyramide Dyes
__
Validated Tyramide Dyes
| __
Incubation Time
| Recommended Starting Concentration| __
Vendor
| __
Catalog #
---|---|---|---|---
CF488A| 8-24 min| 5 µM| Biotium| 92171
CF550R| 8-24 min| 5 µM| Biotium| 96077
CF555| 8-24 min| 5 µM| Biotium| 96021
CF583R| 8-24 min| 5 µM| Biotium| 96085
CF594| 8-24 min| 5 µM| Biotium| 92174
CF640R| 8-24 min| 5 µM| Biotium| 92175
CF754| 8-24 min| 5 µM| Biotium| 96090
Alexa Fluor 488 – Tyramide| 8-24 min| 1x| ThermoFisher|
B40953
Alexa Fluor 546 – Tyramide| 8-24 min| 1x| ThermoFisher|
B40954
Alexa Fluor 647 – Tyramide| 8-24 min| 1x| ThermoFisher|
B40958
Alexa Fluor 750 – Tyramide| 8-24 min| 1x| ThermoFisher|
B56131
Opal 520| 8-24 min| 1:500| Akoya Biosciences|
FP1487001KT
Opal 570| 8-24 min| 1:500| Akoya Biosciences|
FP1488001KT
Opal 620| 8-24 min| 1:500| Akoya Biosciences|
FP1495001KT
Opal 690| 8-24 min| 1:500| Akoya Biosciences|
FP1497001KT
References
- ONCORE Pro Improv Reagent Vials - Biocare Medical
- my.akoyabio.com/ccrz__ProductDetails?sku=FP1487001KT&cclcl=en_US
- my.akoyabio.com/ccrz__ProductDetails?sku=FP1488001KT&cclcl=en_US
- my.akoyabio.com/ccrz__ProductDetails?sku=FP1495001KT&cclcl=en_US
- Tyramide Conjugates
- Tyramide Conjugates
- Tyramide Conjugates
- Tyramide Conjugates