Dec 19, 2024
Multiplexed brightfield detection of CD3e, IBA-1, and Pax5 in formalin-fixed, paraffin-embedded lymph node tissues of pigs, cattle, and white-tailed deer
Forked from a private protocol
- Jayne E Wiarda1,
- Eraldo L Zanella1,
- Mitchell Palmer1
- 1National Animal Disease Center, ARS, USDA
- Jayne Wiarda
Protocol Citation: Jayne E Wiarda, Eraldo L Zanella, Mitchell Palmer 2024. Multiplexed brightfield detection of CD3e, IBA-1, and Pax5 in formalin-fixed, paraffin-embedded lymph node tissues of pigs, cattle, and white-tailed deer. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzk394vx1/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: November 25, 2024
Last Modified: December 19, 2024
Protocol Integer ID: 112726
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
Simultaneous detection of CD3e (T cells; stained in brown), IBA-1 (macrophages/dendritic cells; stained in blue), and Pax5 (B cells; stained in red) in formalin-fixed, paraffin-embedded (FFPE) tracheobronchial lymph node tissues from pigs, cattle, and white-tailed deer.
Attachments
Guidelines
Assay-specific Information
· The general scheme of reagents used to detect each target (IBA-1, CD3e, Pax5) are listed in the table below:
| Antigen retrieval | Protein block | Primary antibody | Secondary antibody | Chromogen/color | |
| None | 2.5% horse serum | Rabbit anti-IBA-1 | Horse anti-rabbit IgG-AP | BCIP/NBT (blue) | |
| Sodium citrate HIER | 2.5% horse serum | Rabbit anti-CD3e | Horse anti-rabbit IgG-HRP | DAB (brown) | |
| Sodium citrate HIER | 2.5% goat serum | Rat anti-Pax5 | Goat anti-rat IgG-AP | Vector Red (red) |
· Primary antibodies are used atspecies-specific dilutions listed in the table below
| Target | Primary antibody | Pig | Cow | Deer | |
| IBA-1 (macrophages/dendritic cells) | Rabbit anti-IBA-1 (stock concentration 500 ug/mL) | 0.5 ug/mL (1:1,000*) 1 hour RT | 0.5 ug/mL (1:1,000*) 1 hour RT | 0.5 ug/mL (1:1,000*) 1 hour RT | |
| CD3e (T cells) | Rabbit anti-CD3e (stock concentration 400 ug/mL) | 0.4 ug/mL (1:1,000*) overnight 4C | 0.4 ug/mL (1:1,000*) overnight 4C | 0.4 ug/mL (1:1,000*) overnight 4C | |
| Pax5 (B cells) | Rat anti-Pax5 (stock concentration 500 ug/mL) | 0.5 ug/mL (1:1,000*) 1 hour RT | 0.5 ug/mL (1:1,000*) 1 hour RT | 0.5 ug/mL (1:1,000*) 1 hour RT |
*Dilution factors will need to be adjusted if stock antibody concentrations differ from those listed in the table
Assay Controls
· Controls will be required to ensure there is minimal/no cross-reactivity with off-target epitopes in the tissue and to ensure stain colors are specific to the intended antibody targets
· All controls should be run on serial sectioned recuts of the same tissue block
· IHC controls:
o No stain control
§ This slide receives only diluent in place of all primary antibodies. All other reagents are still applied to the slide.
o Single antibody stain controls
§ This slide receives only diluent in place of all primary antibodies except one. All other reagents are still applied to the slide.
Assay Variations
· Parameters may need to be further optimized for different 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)
· EZ-Batch Slide Holder (Advanced Cell Diagnostics 321716)
· EZ-Batch Wash Tray (Advanced Cell Diagnostics 321717)
· Tissue-Tek Vertical 24 slide rack (American Master Tech Scientific LWS2124)
· Tissue-Tek Staining Dishes (American Master Tech Scientific LWS20WH)
· Tissue-Tek Clearing Agent Dishes, xylene resistant (American Master Tech Scientific LWS20GR)
· 5.5 Quart Digital Steamer (Hamilton Beach 37530Z)
· Brightfield microscope and/or slide imaging platform
Reagents/Supplies
· Distilled water (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 8668-16)
· 100% ethanol (Pharmco 111000200)
o Dilute with distilled water to make 95%, 85%, and 70% concentrations
· Pro-Par Clearant (Anatech 510)
· Fixative
o 10% NBF (Cancer Diagnostics, Inc. 111) or 4% PFA (Electron Microscopy Sciences 15713)
· ImmEdge Hydrophobic Barrier Pen (Vector H-4000)
· 1X Sodium Citrate Antigen Retrieval Solution, pH 6.0 (made in-house)
o 10X sodium citrate solution: Dissolve 29.4 g sodium citrate dihydrate in 950 mL distilled water. Add water to total volume of 1L after solid has dissolved. Store at 4C up to one year.
o 1X sodium citrate solution: 100 mL 10X sodium citrate solution + 900 mL distilled water. Adjust pH to 6.0. Store at 4C up to three months.
· BLOXALL Endogenous Blocking Solution (Vector SP-6000)
· 1% bovine serum albumin (BSA) in PBS (made in-house)
· 100 mM Tris-HCl with 0.1% Tween, pH 9.5 (made in-house)
· Rabbit anti-IBA-1 polyclonal antibody; stock concentration 500 ug/mL (Fujifilm Wako 019-19741)
o Stock concentration can vary by antibody lot
· Rabbit anti-CD3e polyclonal antibody; stock concentration 0.4 g/mL (Dako A0452)
o Stock concentration can vary by antibody lot
· Rat anti-Pax5 monoclonal antibody, clone 1H9; stock concentration 500 ug/mL (Invitrogen 14-9918-82)
· Horse anti-rabbit IgG-AP polymer (Vector MP-5401)
o Normal Horse Serum 2.5%
o Horse Anti-Rabbit IgG Polymer Reagent
· Horse anti-rabbit IgG-HRP polymer (Vector MP-6401)
o Normal Horse Serum 2.5%
o Horse Anti-Rabbit IgG Polymer Reagent
· Goat anti-rat IgG-AP polymer (Vector MP-5404)
o Normal Goat Serum 2.5%
o Goat Anti-Rat IgG Polymer Reagent
· BCIP/NBT AP substrate (Vector SK-5400)
o BCIP/NBT Reagent 1
o BCIP/NBT Reagent 2
o BCIP/NBT Reagent 3
· DAB HRP substrate (Vector SK-4103)
o DAB EqV Reagent 1 (Chromogen)
o DAB EqV Reagent 2 (Diluent)
· Vector Red AP substrate (Vector SK-5105)
o ImmPACT Red diluent
o ImmPACT Red Reagent 1
o ImmPACT Red Reagent 2
· Gill’s Hematoxylin I (American Master Tech Scientific HXGHE1LT)
· VectaMount PT (Vector H-5600-60)
· #1 thickness coverglass (e.g. Corning 2975-245)
Safety warnings
***For all reagents, refer to MSDS to determine appropriate precautions, personal protective equipment (PPE), and disposal methods before use***
Before start
Starting 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
Before starting the assay:
• Preheat a dry oven to 60℃
• Load slides for assay into vertical slide rack
• Bake slides 20 min 60℃
20m
While slides bake:
o Prepare 0.05% PBS-T (can store at RT up to 1 month)
Immediately before deparaffinizing:
o Add ~200 mL xylenes to each of three clearing agent dishes in a fume hood
o Add ~200 mL 100% ethanol to each of two staining dishes in a fume hood
o Add ~200 mL 95% ethanol to a staining dish in a fume hood
o Add ~200 mL 85% ethanol to a staining dish in a fume hood
o Add ~200 mL 70% ethanol to a staining dish in a fume hood
o Add ~200 mL distilled water to a staining dish in a fume hood
o Add ~200 mL PBS-T to a staining dish
Deparaffinizing & Rehydrating
Deparaffinizing & Rehydrating
25m
25m
o Submerge slide in fresh xylenes 5 min RT
o Submerge slide in fresh xylenes 5 min RT
o Submerge slide in fresh xylenes 5 min RT
o Submerge slides in fresh 100% ethanol 1 min RT
o Submerge slides in fresh 100% ethanol 1 min RT
o Submerge slides in fresh 95% ethanol 1 min RT
o Submerge slides in fresh 85% ethanol 1 min RT
o Submerge slides in fresh 70% ethanol 1 min RT
o Submerge slides in fresh distilled water 3 min RT
o Submerge slides in fresh PBS-T for transport
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 Add ~200 mL PBS-T to each of three staining dishes
o Prepare humidified slide staining tray by adding water to bottom & placing lid on top
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). Slides will remain locked in the EZ-Batch slide holder throughout the protocol until being transferred back to a vertical rack for counterstaining.
• Leave slides in slide staining tray
20m
Tissue Quenching
Tissue Quenching
15m
15m
• Decant slide holder and again place flat in slide staining tray
• Incubate with BLOXALL Endogenous Blocking Solution 10 min 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
15m
While slides incubate with enzyme block:
o Discard deparaffinizing & rehydrating reagents
o Add ~200 mL PBS-T to each of two wash trays
Protein Blocking (horse serum)
Protein Blocking (horse serum)
30m
30m
• Decant slide holder and again place flat in slide staining tray
• Incubate with 2.5% horse serum 30 min RT
o Serum is a component of the horse anti-rabbit IgG-AP polymer kit
o Apply to completely cover tissues; let incubate in slide staining tray with lid closed
30m
While slides incubate with serum:
o Discard tissue quenching reagents
o Prepare diluted anti-IBA-1 antibody by adding IBA-1 antibody stock to 1% BSA in PBS. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. Mix diluted antibody well before use. Dilutions to use are species-specific, as listed in the Antibody Information section at the beginning of this document.
Primary Antibody (IBA-1)
Primary Antibody (IBA-1)
1h 5m
1h 5m
• Decant slide holder and again place flat in slide staining tray
o Do not wash with PBS-T between decanting serum and applying secondary antibody
• Incubate with diluted IBA-1 antibody 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 primary antibody:
o Discard protein blocking reagents
o Add ~200 mL PBS-T to each of two wash trays
Secondary Antibody (anti-rabbit AP)
Secondary Antibody (anti-rabbit AP)
35m
35m
• Decant slide holder and again place flat in slide staining tray
• Incubate with horse anti-rabbit IgG-AP polymer 30 min RT
o Polymer is a component of the horse anti-rabbit IgG-HRP polymer kit
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
While slides incubate with secondary antibody:
o Discard primary antibody reagents
o Add ~200 mL PBS-T to each of two wash trays
Immediately before chromogen detection:
o Prepare diluted BCIP/NBT chromogen by adding two drops of Reagent 1, two drops of Reagent 2, and two drops of Reagent 3 to 5 mL prepared 100mM Tris-HCl in 0.1% Tween, pH 9.5. Total volume to use is dependent on tissue sizes. Make sure to mix reagents thoroughly. Store in the dark due to light sensitivity
Chromogenic Detection (BCIP/NBT)
Chromogenic Detection (BCIP/NBT)
25m
25m
• Decant slide holder and again place flat in slide staining tray
• Incubate with diluted BCIP/NBT chromogen 20 min 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
25m
While slides incubate with chromogen:
o Discard remaining secondary antibody reagents
o Add ~200 mL PBS-T to each of two wash trays
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 Sodium Citrate Buffer, pH 6.0 into staining dish and place in the steamer
o Preheat the prepared steamer, programmed for 1 hour
o Perform this step ~25 minutes before use so that retrieval solution can adequately heat to ~105-110℃
Heat-Induced Epitope Retrieval (HIER)/Antibody Stripping
Heat-Induced Epitope Retrieval (HIER)/Antibody Stripping
20m
20m
• Leave slides in PBS-T at RT until steamer is preheated
• Transfer slides to vertical slide rack
o Do quickly to avoid drying out slides or alternatively place vertical slide rack in a staining dish containing PBS-T and then transfer slides
• Once steamer has preheated, submerge slide rack in preheated 1X Sodium Citrate Buffer 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
• Submerge slide rack in fresh PBS-T 2 min RT
• Submerge slide rack in fresh PBS-T 2 min RT
20m
While slides incubate with sodium citrate:
o Discard chromogen detection reagents
o Add ~200 mL PBS-T to each of two staining dishes
Protein Blocking (horse serum)
Protein Blocking (horse serum)
30m
30m
• Transfer slides back to slide holder
o Do quickly to avoid drying out slides or alternatively place slide holder in a wash tray containing PBS-T and then transfer slides
• Decant slide holder and again place flat in slide staining tray
• Incubate with 2.5% horse serum 30 min RT
o Serum is a component of the horse anti-rabbit IgG-HRP polymer kit
o Apply to completely cover tissues; let incubate in slide staining tray with lid closed
30m
While slides incubate with serum:
o Discard HIER reagents
o Turn off and clean steamer
o Prepare diluted anti-CD3e antibody by adding CD3e antibody stock to 1% BSA in PBS. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. Mix diluted antibody well before use. Dilutions to use are species-specific, as listed in the Antibody Information section at the beginning of this document.
Primary Antibody (CD3e)
Primary Antibody (CD3e)
18h
18h
• Decant slide holder and again place flat in slide staining tray
o Do not wash with PBS-T between decanting serum and applying secondary antibody
• Incubate with diluted CD3e antibody overnight at 4C
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
18h
While slides incubate with primary antibody:
o Discard protein blocking reagents and leftover antibody dilution reagents
The next day:
o Add ~200 mL PBS-T to each of two wash trays
Secondary Antibody (anti-rabbit HRP)
Secondary Antibody (anti-rabbit HRP)
35m
35m
• Decant slide holder and again place flat in slide staining tray
• Incubate with horse anti-rabbit IgG-HRP polymer 30 min RT
o Polymer is a component of the horse anti-rabbit IgG-HRP polymer kit
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
35m
While slides incubate with secondary antibody:
o Discard primary antibody reagents
o Add ~200 mL PBS-T to each of two wash trays
Immediately before chromogen detection:
o Prepare diluted DAB chromogen by adding equal volumes of DAB Reagent 1 (Chromogen) and DAB Reagent 2 (Diluent) together. Total volume to use is dependent on tissue sizes. Make sure to mix reagents thoroughly. Store in the dark due to light sensitivity
Chromogenic Detection (DAB)
Chromogenic Detection (DAB)
15m
15m
• Decant slide holder and again place flat in slide staining tray
• Incubate with diluted DAB chromogen 10 min 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
15m
While slides incubate with chromogen:
o Discard remaining secondary antibody reagents
o Add ~200 mL PBS-T to each of two wash trays
Protein Blocking (goat serum)
Protein Blocking (goat serum)
30m
30m
• Decant slide holder and again place flat in slide staining tray
• Incubate with 2.5% goat serum 30 min RT
o Serum is a component of the goat anti-rat IgG-AP polymer kit
o Apply to completely cover tissues; let incubate in slide staining tray with lid closed
• Decant slides and transfer to vertical slide rack
30m
While slides incubate with serum:
o Discard chromogen detection reagents
o Prepare diluted anti-Pax5 antibody by adding Pax5 antibody stock to 1% BSA in PBS. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. Mix diluted antibody well before use. Dilutions to use are species-specific, as listed in the Antibody Information section at the beginning of this document.
Primary Antibody (Pax5)
Primary Antibody (Pax5)
1h 5m
1h 5m
• Decant slide holder and again place flat in slide staining tray
o Do not wash with PBS-T between decanting serum and applying secondary antibody
• Incubate with diluted Pax5 antibody 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 primary antibody:
o Discard protein blocking reagents and leftover antibody dilution reagents
o Add ~200 mL PBS-T to each of two wash trays
Secondary Antibody (anti-rat AP)
Secondary Antibody (anti-rat AP)
35m
35m
• Decant slide holder and again place flat in slide staining tray
• Incubate with goat anti-rat IgG-AP polymer 30 min RT
o Polymer is a component of the goat anti-rat IgG-AP polymer kit
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
35m
While slides incubate with secondary antibody:
o Discard primary antibody reagents
o Add ~200 mL PBS-T to each of two wash trays
Immediately before chromogen detection:
o Prepare diluted Vector Red chromogen by adding 2 drops of Reagent 1 and two drops of Reagent 2 to 5 mL Diluent. Total volume to use is dependent on tissue sizes. Make sure to mix reagents thoroughly. Store in the dark due to light sensitivity
Chromogenic Detection (Vector Red)
Chromogenic Detection (Vector Red)
35m
35m
• Decant slide holder and again place flat in slide staining tray
• Incubate with diluted Vector Red chromogen 30 min 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
35m
While slides incubate with chromogen:
o Discard secondary antibody reagents
o Add ~200 mL PBS-T to each of two wash trays
o Add ~200 mL hematoxylin to one staining dish
o Add ~200 mL distilled water to each of three staining dishes
Counterstaining
Counterstaining
2m
2m
• Transfer slides to vertical slide rack
o Do quickly to avoid drying out slides or alternatively place vertical slide rack in a staining dish containing PBS-T and then transfer slides
• Submerge slide rack in hematoxylin 1 min RT
• Submerge slide rack in fresh distilled water, dunking 3-5 times
• Submerge slide rack in fresh distilled water, dunking 3-5 times
• Submerge slide rack in fresh distilled water, dunking 3-5 times
o Water should no longer appear purple in the third water dish used
2m
Mounting
Mounting
45m
45m
• Air dry slides RT 20-30 min
• In a fume hood, mount slides by adding 2-4 drops of VectaMount mounting media to each slide, followed by application of a cover glass. Remove bubbles from tissue by applying pressure to cover glass
• Place slides flat in a dry, dark space to air dry at RT overnight
• Assess staining with a bright-field microscope
45m
Protocol references
Contributions/Acknowledgements
· Staining protocol was developed by Dr. Jayne Wiarda
· Staining protocol was optimized and executed by Dr. Eraldo Zanella
· We thank Colin Stoy for additional wet lab assistance
· We thank Judith Stasko and Adrienne Shircliff for slide sectioning and image scanning
· We thank Dr. Mitchell Palmer for providing archived tissue specimens