Dec 19, 2024
Co-detection of RNA and protein (CD3, IBA-1, Pax5, Ki-67, or pan-cytokeratin type I/II) in formalin-fixed, paraffin-embedded tonsil tissues of pigs, cattle, and white-tailed deer
Forked from a private protocol
- Jayne E Wiarda1,
- adrienne.shircliff1,
- Eraldo Zanella1,
- Crystal L Loving1,
- Mitchell Palmer1
- 1National Animal Disease Center, ARS, USDA
- Jayne Wiarda
Protocol Citation: Jayne E Wiarda, adrienne.shircliff, Eraldo Zanella, Crystal L Loving, Mitchell Palmer 2024. Co-detection of RNA and protein (CD3, IBA-1, Pax5, Ki-67, or pan-cytokeratin type I/II) in formalin-fixed, paraffin-embedded tonsil tissues of pigs, cattle, and white-tailed deer. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldr5o8g5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 19, 2024
Last Modified: December 19, 2024
Protocol Integer ID: 116335
Funders Acknowledgements:
USDA-ARS
Grant ID: CRIS #5030-32000-230-000-D
Disclaimer
All opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of USDA, ARS, DOE, or ORAU/ORISE. Mention of trade names or products is for information purposes only and does not imply endorsement by the USDA. USDA is an equal opportunity employer and provider.
Abstract
Detection of CD3e (T cells), IBA-1 (macrophages/dendritic cells), Pax5 (B cells), Ki-67 (proliferating cells), or pan-cytokeratin type I/II (epithelial cells) in conjunction with an RNA marker (housekeeping gene, UBC, used herein) in formalin-fixed, paraffin-embedded (FFPE) palatine tonsil tissues from pigs, cattle, and white-tailed deer.
Attachments
Guidelines
Assay-specific Information
· Reagents for primary and secondary antibodies are specific to the target being detected. The general scheme of these reagents used to detect each specific target are listed in the table below.
· Primary antibodies are used at species-specific dilutions and incubation times listed in the table below.
Assay Controls
· Controls will be required to ensure there is minimal/no cross-reactivity with off-target epitopes in the tissue and to ensure stains are specific to the intended antibody targets
· To create a negative control slide, antibody diluent should be applied in place of primary antibody, and the DapB RNAscope probe should be applied to slides. All other reagents are still applied to the slide as normal.
Assay Variations
· Parameters may need to be further optimized for different experiments, tissues, targets, or species.
Materials
Equipment
· Pipettes/pipette tips
· Dry weigh scale (0.1 gram increments required)
· Weigh boats
· Reagent weighing spatulas
· pH meter
· Graduated cylinders for liquid measurements (1 mL increments required)
· Glassware/plasticware for liquid reagents
· Drying oven (able to reach & hold 60℃)
· Fume hood
· Slide staining tray (e.g. Simport M920-2)
· Tissue-Tek Vertical 24 slide rack (Tissue Tech LWS2124)
· Tissue-Tek Staining Dishes (Tissue Tech LWS20WH)
· Tissue-Tek Clearing Agent Dishes, xylene resistant (Tissue Tech LWS20GR)
· HybEZ II Hybridization System with EZ-Batch Slide System (Advanced Cell Diagnostics 321710/321720)
o HybEZ oven (Advanced Cell Diagnostics 321710/321720)
o Humidity control tray (Advanced Cell Diagnostics 310012)
o HybEZ Humidifying Paper (Advanced Cell Diagnostics 310025)
o EZ-Batch Wash Tray (Advanced Cell Diagnostics 321717)
o EZ-Batch Slide Holder (Advanced Cell Diagnostics 321716)
· 5.5 Quart Digital Steamer (Hamilton Beach 37530Z)
· Confocal/fluorescent microscope and/or slide imaging platform
Reagents/Supplies
· Distilled waterc(obtained in-house)
· Phosphate-buffered saline (PBS), pH 7.2-7.4 (made in-house)
· 0.05% PBS-Tween (PBS-T), pH 7.35 (made in-house)
· Xylenes (Macron Fine Chemicals 8668-06)
· 100% ethanol (Pharmco 111000200)
· Fixative
o 10% NBF (Cancer Diagnostics, Inc. 111) or 4% PFA (Electron Microscopy Sciences 15713)
· ImmEdge Hydrophobic Barrier Pen (Vector H-4000)
· SSC buffer (ThermoFisher Scientific J60839.K3)
· RNAscope H2O2 & Protease Plus Reagents (Advanced Cell Diagnostics 322330/322381)
o Hydrogen Peroxide (Advanced Cell Diagnostics 322335)
o Protease Plus (Advanced Cell Diagnostics 322337)
· RNA-Protein Co-Detection Ancillary Kit (Advanced Cell Diagnostics 323180)
o Co-Detection Target Retrieval Reagents (Advanced Cell Diagnostics 322000)
o Co-Detection Antibody Diluent (Advanced Cell Diagnostics 323160)
o Co-Detection Blocker (Advanced Cell Diagnostics 323170)
· RNAscope Wash BufferReagents (Advanced Cell Diagnostics 310091/320058)
· RNAscope Multiplex Fluorescent Detection Reagents v2 (Advanced Cell Diagnostics 323110)
o AMP 1 (Advanced Cell Diagnostics 323101)
o AMP 2 (Advanced Cell Diagnostics 323102)
o AMP 3 (Advanced Cell Diagnostics 323103)
o HRP-C1 (Advanced Cell Diagnostics 323104)
o HRP blocker (Advanced Cell Diagnostics 323107)
· RNAscope channel 1 probe (interchangeable with other RNAscope probes)
o Housekeeping gene: H.s. UBC (Advanced Cell Diagnostics 310041)
o Negative control: DapB (Advanced Cell Diagnostics 310043)
· Opal 570 fluorophore (Akoya OP001003)
· TSA 650 fluorophore (Advanced Cell Diagnostics 323273)
· Rabbit anti-CD3e polyclonal antibody; stock concentration 0.4 g/mL (Dako A0452)
o Stock concentration can vary by antibody lot
· Rabbit anti-IBA-1 polyclonal antibody; stock concentration 500 ug/mL (Fujifilm Wako 019-19741)
o Stock concentration can vary by antibody lot
· Goat anti-IBA-1 polyclonal antibody; stock concentration 600 ug/mL (Fujifilm Wako 011-27991)
o Stock concentration can vary by antibody lot
· Mouse anti-Ki-67 monoclonal antibody, clone B56; stock concentration 250 ug/mL (BD Biosciences 550609)
· Mouse anti-cytokeratin type I/II clonal antibody, clones AE1/AE3; stock concentration 200 ug/mL (Novus Biologicals NBP2-29429)
· Rat anti-Pax5 monoclonal antibody, clone 1H9; stock concentration 500 ug/mL (Invitrogen 14-9918-82)
· Horse anti-rabbit IgG-HRP polymer (Vector MP-6401)
o Horse Anti-Rabbit IgG Polymer Reagent
· Horse anti-goat IgG-HRP polymer (Vector MP-7405)
o Horse Anti-Goat IgG Polymer Reagent
· Horse anti-mouse IgG-HRP polymer (Vector MP-6402)
o Horse Anti-Mouse IgG Polymer Reagent
· Goat anti-rat IgG-HRP polymer (Vector MP-7404)
o Goat Anti-Rat IgG Polymer Reagent
· ProLong Gold mounting media with DAPI (Invitrogen P36931)
· #1.5 thickness cover glass (e.g. Fisherbrand 2980-245)
Safety warnings
***For all reagents, refer to MSDS to determine appropriate precautions, personal protective equipment (PPE), and disposal methods before use***
Before start
Starting specimens
Starting samples = FFPE tissues cut to 4 micron thickness and adhered to positively-charged microscopy slides (e.g. SuperFrost Plus Slides; Fisher Scientific 12-550-15). It is crucial that tissues are adequately fixed to prevent tissue degradation. Tissues no thicker than 0.5 centimeters should be freshly harvested and placed into 10% neutral-buffered formalin (NBF) or 4% paraformaldehyde (PFA) at a ratio of at least 20 volumes fixative per one volume tissue. Fix tissues between 16-30 hours at room temperature (RT), followed by immediate transfer to 70% ethanol and processing into FFPE tissue blocks. Fixation times should be optimized for individual tissues and experiments.
Baking
Baking
30m
30m
Before starting the assay:
• Preheat a dry oven to 60℃
• Load slides for assay into vertical slide rack
• Bake slides 30 min 60℃
30m
While slides bake:
o Prepare 0.05% PBS-T
Immediately before deparaffinizing:
o Add ~200 mL xylenes to each of two clearing agent dishes in a fume hood
o Add ~200 mL 100% ethanol to each of two staining dishes in a fume hood
Deparaffinizing & Rehydrating
Deparaffinizing & Rehydrating
25m
25m
• Submerge slide rack in fresh xylenes 5 min RT
• Submerge slide rack in fresh xylenes 5 min RT
• Submerge slide rack in fresh 100% ethanol 5 min RT
• Submerge slide rack in fresh 100% ethanol 5 min RT
• Air dry slides ~5 min or until completely dry
25m
While slides deparaffinize/rehydrate:
o Turn off dry oven
o Prepare humidified slide staining tray by adding water to bottom & placing lid on top
o Prepare 1X Co-detection Target Retrieval solution by adding one bottle (70 mL) 10X co-detection target retrieval solution to 630 mL distilled water. 1X solution is good for up to one month stored at 4C.
o Prepare steamer:
o Fill the bottom reservoir of steamer to the ‘Fill’ line with tap water
o Assemble the steamer, using both tiers 1 and 2 of steam bowls. Do not place the divider between steam bowls, as an extra tall compartment is needed
o Pour 200 mL prepared 1X Co-detection Target Retrieval solution into staining dish and place in the steamer
o Preheat the prepared steamer, programmed for 1 hour
o Perform this step ~25-30 minutes before use so that retrieval solution can adequately heat to ~105-110℃
Tissue Quenching
Tissue Quenching
11m
11m
• Remove dried slides from vertical rack and place slides flat inside the slide staining tray
• Incubate with Hydrogen Peroxide 10 min RT
o Apply to completely cover tissues; let incubate in slide staining tray with lid closed
• Decant slides and transfer to vertical slide rack
• Submerge slide rack in fresh distilled water, dunking 3-5 times
• Submerge slide rack in fresh distilled water, dunking 3-5 times
11m
While slides incubate with hydrogen peroxide:
o Discard deparaffinizing & rehydrating reagents
o Add ~200 mL distilled water to each of two staining dishes
Heat-Induced Epitope Retrieval (HIER)
Heat-Induced Epitope Retrieval (HIER)
16m
16m
• Leave slide rack in water at RT until steamer is preheated
• Once steamer has preheated, submerge slide rack in preheated 1X Co-detection Target Retrieval solution 15 min
o Slides should be maintained at ~105-110℃ for the duration of target retrieval. To ensure temperature is not reduced, open steamer lid, load slides, and shut steamer lid as quickly as possible.
• Remove slide rack from steamer and turn off steamer
• Submerge slide rack in fresh distilled water, dunking 3-5 times
• Submerge slide rack in fresh PBS-T, dunking 3-5 times
• Leave slides submerged in PBS-T
16m
While slides incubate with target retrieval solution:
o Discard tissue quenching reagents
o Add ~200 mL distilled water to one staining dish
o Add ~200 mL PBS-T to one staining dish
o Prepare diluted primary antibody by adding antibody stock to co-detection antibody diluent. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. Mix diluted antibody well before use. Refer to assay-specific information above to determine the appropriate antibody dilution to use.
Hydrophobic Barrier
Hydrophobic Barrier
20m
20m
• Apply hydrophobic barrier around each tissue
o One by one, unload slides from vertical rack submerged in PBS-T. Dry off only the area around the tissue where a barrier will be drawn with a hydrophobic barrier pen. Keep tissue area wet the whole time. Draw barrier and place slides into the EZ-Batch slide holder placed inside the slide staining tray. Using a pipette, apply a small amount of PBS-T within the barrier (just enough to keep the tissue wet while drawing barriers on remaining slides).
• Leave slide holder in slide staining tray
20m
Primary Antibody
Primary Antibody
18h
18h
• Decant slide holder and again place flat in slide staining tray
• Incubate with diluted primary antibody 1 hour RT or 4C overnight
o Refer to assay-specific information above to determine antibody incubation time
o Apply to completely cover tissues; let incubate in slide staining tray with lid closed
• Decant slide holder and transfer to wash trays for PBS-T washes
• Submerge slide holder in fresh PBS-T 2 min RT
• Submerge slide holder in fresh PBS-T 2 min RT
• Submerge slide holder in fresh PBS-T 2 min RT
1h 6m
Optional stopping point #1
Optional stopping point #1
18h
18h
While slides incubate with primary antibody:
o Prepare 5X SSC buffer by adding 15 mL 20X SSC buffer to 285 mL dH2O and placing into a wash tray
o Adjust additional preparations outlined during primary antibody incubation below to also accommodate needs/timeline
• Submerge slide holder in 5X SSC buffer overnight RT
• Decant slide holder and transfer to wash trays for PBS-T washes
• Submerge slide holder in fresh PBS-T 2 min RT
• Submerge slide holder in fresh PBS-T 2 min RT
18h
Antibody Crosslinking
Antibody Crosslinking
36m
36m
While slides incubate with primary antibody:
o Discard HIER reagents
o Add ~200 mL PBS-T to each of three wash trays
o Add ~200 mL 10% neutral-buffered formalin to one vertical slide staining dish in a fume hood
o Prepare the HybEZ oven:
o Place a humidifying paper inside the humidity control tray of the HybEZ oven. Turn the oven on and set to 40C to preheat with the humidifying tray inside the oven. Preheat the oven at least 30 minutes prior to use.
• Decant slide holder and transfer to vertical slide staining rack for formalin fixation
• Submerge slide holder in fresh 10% neutral-buffered formalin 30 min RT
• Decant slide rack and transfer to staining dishes for PBS-T washes
• Submerge slide rack in fresh PBS-T 2 min RT
• Submerge slide rack in fresh PBS-T 2 min RT
• Submerge slide rack in fresh PBS-T 2 min RT
36m
While slides incubate with formalin:
o Discard primary antibody reagents
o Add ~200 mL PBS-T to each of three staining dishes
Protease Digestion
Protease Digestion
16m
16m
• Transfer slides from vertical slide rack back into slide holder and place in humidifying tray taken from preheated HybEZ oven
• Incubate with Protease Plus 15 min 40C
o Apply to completely cover tissues; let incubate in humidifying tray placed within preheated HybEZ oven
• Remove slide holder from HybEZ oven, decant, and transfer to wash trays for PBS-T washes
• Submerge slide holder in fresh distilled water, dunking 3-5 times
• Submerge slide holder in fresh distilled water, dunking 3-5 times
16m
While slides incubate with protease:
o Discard antibody crosslinking reagents
o Add ~200 mL distilled water to each of two wash trays
o Preheat RNAscope probe bottles to 40℃ for 10 min before use by placing them inside the HybEZ oven during protease incubation. Total volume to use is dependent on tissue sizes.
Probe Hybridization
Probe Hybridization
2h 4m
2h 4m
• Decant slide holder and place in humidifying tray taken from preheated HybEZ oven
• Incubate with prewarmed, undiluted RNAscope probe 2 hours 40C
o Invert bottle immediately before use; apply to completely cover tissues; let incubate in humidifying tray placed within preheated HybEZ oven
• Remove slide holder from HybEZ oven, decant, and transfer to wash trays for buffer washes
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
2h 4m
Optional stopping point #2
Optional stopping point #2
18m
18m
While slides incubate with probe:
o Prepare 5X SSC buffer by adding 15 mL 20X SSC buffer to 285 mL dH2O and placing into a wash tray
o Adjust additional preparations outlined during primary antibody incubation below to also accommodate needs/timeline
• Submerge slide holder in 5X SSC buffer overnight RT
• Decant slide holder and transfer to wash trays for PBS-T washes
• Submerge slide holder in fresh PBS-T 2 min RT
• Submerge slide holder in fresh PBS-T 2 min RT
18m
RNA Detection
RNA Detection
2h 45m
2h 45m
While slides incubate with probe:
o Discard protease digestion reagents
o Prepare 1X Wash Buffer:
o Add two 10X bottles (2x60 mL) of wash buffer to 5.88 L of distilled water. 1X solution is stable at RT up to one month.
o Add ~200 mL wash buffer to each of two wash trays
o Place remaining RNAscope reagents at RT at least 30 min before use
• Decant slide holder and place in humidifying tray taken from preheated HybEZ oven
• Incubate with AMP1 30 min 40C
o Invert bottle immediately before use; apply to completely cover tissues; let incubate in humidifying tray placed within preheated HybEZ oven
• Remove slide holder from HybEZ oven, decant, and transfer to wash trays for buffer washes
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
• Decant slide holder and place in humidifying tray taken from preheated HybEZ oven
• Incubate with AMP2 30 min 40C
o Invert bottle immediately before use; apply to completely cover tissues; let incubate in humidifying tray placed within preheated HybEZ oven
• Remove slide holder from HybEZ oven, decant, and transfer to wash trays for buffer washes
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
• Decant slide holder and place in humidifying tray taken from preheated HybEZ oven
• Incubate with AMP3 15 min 40C
o Invert bottle immediately before use; apply to completely cover tissues; let incubate in humidifying tray placed within preheated HybEZ oven
• Remove slide holder from HybEZ oven, decant, and transfer to wash trays for buffer washes
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
• Decant slide holder and place in humidifying tray taken from preheated HybEZ oven
• Incubate with HRP-C1 15 min 40C
o Invert bottle immediately before use; apply to completely cover tissues; let incubate in humidifying tray placed within preheated HybEZ oven
• Remove slide holder from HybEZ oven, decant, and transfer to wash trays for buffer washes
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
• Decant slide holder and place in humidifying tray taken from preheated HybEZ oven
• Incubate with diluted Opal 570 30 min 40C
o Invert bottle immediately before use; apply to completely cover tissues; let incubate in humidifying tray placed within preheated HybEZ oven
• Remove slide holder from HybEZ oven, decant, and transfer to wash trays for buffer washes
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
• Decant slide holder and place in humidifying tray taken from preheated HybEZ oven
• Incubate with HRP blocker 15 min 40C
o Invert bottle immediately before use; apply to completely cover tissues; let incubate in humidifying tray placed within preheated HybEZ oven
• Remove slide holder from HybEZ oven, decant, and transfer to wash trays for buffer washes
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
• Submerge slide holder in fresh 1X Wash Buffer 2 min RT
2h 45m
Immediately before Opal incubation:
o Prepare diluted Opal fluorophore by diluting Opal 570 into Multiplex TSA Buffer at a dilution of 1:750. Total volume to use is dependent on tissue sizes. Make sure to mix reagents thoroughly. Store in the dark due to light sensitivity.
During each incubation:
o Discard reagents from previous incubation step
o Add ~200 mL 1X wash buffer to each of two wash trays
While slides incubate with blocker:
o Discard remaining RNA detection reagents
o Add ~200 mL wash buffer to each of two wash trays
Secondary Antibody
Secondary Antibody
35m
35m
• Decant slide holder and again place flat in slide staining tray
• Incubate with secondary antibody (HRP-conjugated polymer) 30 min RT
o Refer to assay-specific information above to determine which secondary antibody to use
o Secondary antibody polymer is a component of the secondary antibody reagent kit
o Invert bottle immediately before use; apply to completely cover tissues; let incubate in slide staining tray with lid closed
• Decant slide holder and transfer to wash trays for PBS-T washes
• Submerge slide holder in fresh PBS-T 2 min RT
• Submerge slide holder in fresh PBS-T 2 min RT
35m
While slides incubate with secondary antibody:
o Discard tissue blocking reagents
o Add ~200 mL PBS-T to each of two wash trays
o Turn off HybEZ oven
Immediately before protein detection:
o Prepare diluted TSA fluorophore by diluting TSA 650 into Multiplex TSA Buffer at a dilution of 1:750. Total volume to use is dependent on tissue sizes. Make sure to mix reagents thoroughly. Store in the dark due to light sensitivity.
Protein Detection
Protein Detection
1h 5m
1h 5m
• Decant slide holder and again place flat in slide staining tray
• Incubate with diluted TSA 650 1 hour RT
o Invert bottle immediately before use; apply to completely cover tissues; let incubate in humidifying tray placed within preheated HybEZ oven
• Decant slide holder and transfer to wash trays for PBS-T washes
• Submerge slide holder in fresh PBS-T 2 min RT
• Submerge slide holder in fresh PBS-T 2 min RT
1h 5m
While slides incubate with TSA:
o Discard secondary antibody reagents
o Add ~200 mL PBS-T to each of two wash trays
Nuclei Staining and Coverslipping
Nuclei Staining and Coverslipping
1h
1h
• Mount slides by adding 2-4 drops of ProLong Gold mounting media containing DAPI to each slide, followed by application of a #1.5 thickness cover glass. Remove bubbles from tissue by applying pressure to cover glass.
• Place slides flat in a dry, dark space to air dry 30 min RT
• Transfer to 4C and image with a fluorescent or confocal microscope within two weeks
1h
While slides are air drying:
o Discard protein detection reagents
Protocol references
Contributions/Acknowledgements
· Staining protocol was developed by Dr. Jayne Wiarda
· Staining protocol was optimized and executed by Dr. Jayne Wiarda, Dr. Eraldo Zanella, and Colin Stoy
· We thank Adrienne Shircliff for slide sectioning and imaging
· We thank Drs. Mitchell Palmer and Crystal Loving for providing archived tissue specimens