Dec 19, 2024
CD3e protein, Pax5 protein, and Senecavirus RNA detection in formalin-fixed, paraffin-embedded (FFPE) pig tissues
Forked from a private protocol
- Jayne E Wiarda1,
- adrienne.shircliff1,
- Eric Cassmann1,
- Alexandra Buckley1
- 1National Animal Disease Center, ARS, USDA
- Jayne Wiarda
Protocol Citation: Jayne E Wiarda, adrienne.shircliff, Eric Cassmann, Alexandra Buckley 2024. CD3e protein, Pax5 protein, and Senecavirus RNA detection in formalin-fixed, paraffin-embedded (FFPE) pig tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp9d4dvzp/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: 116337
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) and Pax5 (B cells) in conjunction with Senecavirus A (SVA) viral capsid RNA in formalin-fixed, paraffin-embedded (FFPE) palatine tonsil tissue from a pig experimentally infected with SVA.
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Materials
Equipment
· Pipettes/pipette tips – volumes ranging between 2-1000 uL
· Drying oven (able to reach & hold 60℃)
· Fume hood
· Slide staining tray (e.g. Simport M920-2)
· 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)
· 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)
· 5.5 Quart Digital Steamer (Hamilton Beach 37530Z)
· Confocal/fluorescent microscope and/or slide imaging platform
Reagents/Supplies
· Distilled water (obtained in-house)
· 0.05% PBS-Tween (PBS-T), pH 7.35 (made in-house)
· Xylenes (Macron Fine Chemicals 8668-16)
· 100% ethanol (Pharmco 111000200)
· 10% NBF (3.7% formaldehyde; Cancer Diagnostics, Inc. 111)
· ImmEdge Hydrophobic Barrier Pen (Vector H-4000)
· 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 Buffer Reagents (Advanced Cell Diagnostics 310091/320058)
· RNAscope Probes, Channel 1 (interchangeable with other channel 1 probes if desiring to target other transcripts)
o V-SVV-VP (Advanced Cell Diagnostics 450221)
• Reactive to Senecavirus A nucleocapsid gene sequence
· 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 Multiplex TSA Buffer (ACD 322809)
· Opal 570 (Akoya OP-001003)
· Polyclonal Rabbit Anti-human CD3e antibody (Dako A0452, stock concentration 0.4 g/L)
· Monocolonal Rat Pax5 antibody (Invitrogen 14-9918-82 0.5mg/ml)
· Donkey anti-rabbit IgG AF647 (Invitrogen A31573)
· Donkey anti-anti-rat IgG AF488 (Invitrogen A20208)
· 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
30m
· Bake slides 30 min 60℃
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
o Submerge slide rack in fresh xylenes 5 min RT
o Submerge slide rack in fresh xylenes 5 min RT
o Submerge slide rack in fresh 100% ethanol 5 min RT
o Submerge slide rack in fresh 100% ethanol 5 min RT
o 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:
· Discard tissue quenching reagents
· Add ~200 mL distilled water to one staining dish
· Add ~200 mL PBS-T to one staining dish
- Add antibodies for overnight incubation to Co-Detection Antibody Diluent at dilutions as follows: Pax5 1:100 and CD3e 1:400. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting.
Hydrophobic Barrier
Hydrophobic Barrier
25m
25m
· 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
25m
Primary Antibody
Primary Antibody
18h
18h
· Decant slide holder and again place flat in slide staining tray
· Incubate with diluted primary antibody cocktail 4C overnight
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
18h
While slides are incubating with primary antibodies:
o Discard HIER reagents
o Add ~200 mL PBS-T to each of three to each of three wash trays
o Add ~200 mL 10% neutral-buffered formalin to one vertical slide staining tray 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.
Antibody Crosslinking
Antibody Crosslinking
36m
36m
· Decant slide holder and transfer to vertical slide staining rack for formalin fixation
· Submerge slide rack 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 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 bottle 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 Senecavirus 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 WashBuffer 2 min RT
· Submerge slide holder in fresh 1X Wash Buffer 2 min RT
2h 4m
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
RNA Detection
RNA Detection
2h 45m
2h 45m
· 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:
- Discard reagents from previous incubation step
- Add ~200 mL 1X wash buffer to each of two wash trays
While slides incubate with HRP blocker:
o Discard remaining RNA detection reagents
o Add ~200 mL wash buffer to each of two wash trays
o Prepare diluted secondary antibody by diluting into Co-detection Antibody Diluent as follows: donkey anti-rabbit AF647 1:400 and donkey anti-rat AF488 1:200. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. Store in the dark due to light sensitivity.
Secondary Antibody/Protein Detection
Secondary Antibody/Protein Detection
1h 5m
1h 5m
· Decant slide holder and again place flat in slide staining tray
· Incubate with diluted secondary antibody cocktail 1 hour RT
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
1h 5m
While slides incubate with secondary antibody:
o Discard HRP blocker reagents
o Add ~200 mL PBS-T to each of two wash trays
o Turn off HybEZ oven
Nuclei Staining and Coverslipping
Nuclei Staining and Coverslipping
50m
50m
· 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
50m
While slides are air drying:
· Discard secondary antibody/protein detection reagents
Protocol references
Staining protocol was developed by Dr. Jayne Wiarda
Staining protocol was optimized and executed by Dr. Jayne Wiarda and Colin Stoy
We thank Adrienne Shircliff for slide sectioning and imaging
We thank Drs. Eric Cassman and Alexandra Buckley for providing archived tissue specimens