Dec 19, 2024
Multiplexed fluorescent detection of CD3e, IBA-1, Pax5, and Ki-67 in formalin-fixed, paraffin-embedded pig lymph node tissue
Forked from a private protocol
- Jayne E Wiarda1,
- adrienne.shircliff1,
- Eraldo L Zanella1
- 1National Animal Disease Center, ARS, USDA
- Jayne Wiarda
Protocol Citation: Jayne E Wiarda, adrienne.shircliff, Eraldo L Zanella 2024. Multiplexed fluorescent detection of CD3e, IBA-1, Pax5, and Ki-67 in formalin-fixed, paraffin-embedded pig lymph node tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk94yxv5r/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: 112725
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), IBA-1 (macrophages/dendritic cells), Pax5 (B cells), and Ki-67 (proliferating cells) in formalin-fixed, paraffin-embedded (FFPE) pig lymph node tissue
Attachments
Guidelines
Antibody Dilutions/Incubations
· All antibodies are diluted in 1% BSA in PBS
· Primary antibodies anti-CD3e, Pax5, and Ki-67 are combined into a single cocktail when diluted; anti IBA-1 antibody is diluted in a separate mixture
· All secondary antibodies are combined into a single cocktail when diluted
| Primary antibody | Dilution/Incubation | Secondary antibody | Dilution/Incubation | |
| Rabbit anti-CD3e (stock concentration 400 ug/mL) | 0.4 ug/mL (1:1,000*) overnight 4C | Anti-rabbit IgG-AF546 (stock concentration 2,000 ug/mL) | 5 ug/mL (1:400*) 1 hour RT | |
| Rat anti-Pax5 (stock concentration 500 ug/mL) | 0.05 ug/mL (1:10,000*) overnight 4C | Anti-rat IgG-AF594 (stock concentration 2,000 ug/mL) | 20 ug/mL (1:100*) 1 hour RT | |
| Mouse anti-Ki-67 (stock concentration 250 ug/mL) | 0.025 ug/mL (1:10,000*) overnight 4C | Anti-mouse IgG-AF488 (stock concentration 2,000 ug/mL) | 20 ug/mL (1:100*) 1 hour RT | |
| Goat anti-IBA-1 (stock concentration 600 ug/mL) | 0.3 ug/mL (1:2,000*) 1 hour RT | Anti-goat IgG-AF647 (stock concentration 2,000 ug/mL) | 8 ug/mL (1:250*) 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 properly set parameters for spectral demultiplexing during slide imaging
· 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 secondary antibodies are still applied to the slide. Mounting media that does not contain DAPI is used for slide mounting.
o Single antibody stain controls
§ This slide receives only diluent in place of all primary antibodies except one. All secondary antibodies are still applied to the slide. Mounting media that does not contain DAPI is used for slide mounting.
o DAPI only control
§ This slide receives only diluent in place of all primary antibodies. All secondary antibodies are still applied to the slide. Mounting media that contains DAPI is used for slide mounting.
o Full stain control
§ All primary and secondary antibodies are applied to this slide. Mounting media that contains DAPI is used for slide mounting.
· Controls for this assay include the following 7 controls: (1) no stain; (2) CD3e single stain; (3) IBA-1 single stain; (4) Pax5 single stain; (5) Ki-67 single stain; (6) DAPI only; and (7) full stain
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)
· Confocal microscope
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)
· Normal donkey serum (Abcam 7475)
o Aliquot and store at -20C; avoid freeze-thaw cycles
o Used at working concentration of 5%, diluted in PBS
· Rabbit anti-CD3e polyclonal antibody; stock concentration 0.4 g/mL (Dako A0452)
o Stock concentration can vary by antibody lot
· Goat anti-IBA-1 polyclonal antibody; stock concentration 0.6 mg/mL (Fujifilm Wako 011-27991)
o Stock concentration can vary by antibody lot
· Rat anti-Pax5 monoclonal antibody, clone 1H9; stock concentration 500 ug/mL (Invitrogen 14-9918-82)
· Mouse anti-Ki-67 monoclonal antibody, clone B56; stock concentration 250 ug/mL (BD 550609)
· Donkey anti-rabbit IgG polyclonal antibody, conjugated to Alexa Fluor 546; stock concentration 2 mg/mL (Invitrogen A10040)
· Donkey anti-goat IgG polyclonal antibody, conjugated to Alexa Fluor 647; stock concentration 2 mg/mL (Invitrogen A21447)
· Donkey anti-Rat IgG polyclonal antibody, conjugated to Alexa Fluor 594; stock concentration 2 mg/mL (Invitrogen A21209)
· Donkey anti-mouse IgG polyclonal antibody, conjugated to Alexa Fluor 488; stock concentration 2 mg/mL (Invitrogen A21202)
· Pro-Long Gold with DAPI mounting media (Invitrogen P3693)
· Pro-Long Gold Mounting media (Invitrogen P36930)
o This item does NOT contain DAPI
· #1.5 thickness cover glass (e.g. Corning 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
20m
20m
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
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 rack in fresh xylenes 5 min RT
o Submerge slide rack in fresh xylenes 5 min RT
o Submerge slide rack in fresh xylenes 5 min RT
o Submerge slides rack in fresh 100% ethanol 1 min RT
o Submerge slides rack in fresh 100% ethanol 1 min RT
o Submerge slides rack in fresh 95% ethanol 1 min RT
o Submerge slides rack in fresh 85% ethanol 1 min RT
o Submerge slides rack in fresh 70% ethanol 1 min RT
o Submerge slides rack in fresh distilled water 3 min RT
o Submerge slides rack 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 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 ~25-30 minutes before use so that retrieval solution can adequately heat to ~105-110℃
Heat-Induced Epitope Retrieval (HIER)
Heat-Induced Epitope Retrieval (HIER)
20m
20m
• Leave slide rack in PBS-T at RT until steamer is preheated
• 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 and turn off 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 buffer:
o Discard deparaffinizing & rehydrating reagents
o Add ~200 mL PBS-T to each of two staining dishes
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 slide holder 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 HIER reagents
o Double check steamer is turned off
o Add ~200 mL PBS-T to each of two wash trays
o Thaw aliquot of donkey serum and dilute to 5% in PBS (1 volume serum per 19 volumes PBS)
Protein Blocking
Protein Blocking
· Decant slide holder and again place flat in slide staining tray
· Incubate with 5% donkey serum 30 min RT
o Apply to completely cover tissues; let incubate in slide staining tray with lid closed
While slides incubate with serum:
o Discard tissue quenching reagents
o Prepare cocktail of primary antibodies incubated overnight (anti-CD3e, Pax5, Ki-67) by adding antibodies to 1% BSA in PBS. Refer to antibody dilutions/incubations above to determine the appropriate antibody dilution to use. Also refer to assay controls information for any slides that should not receive antibody. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. Mix diluted antibody well before use.
Primary Antibody
Primary Antibody
19h
19h
• 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 primary antibodies (anti-CD3e, Pax5, Ki-67) overnight at 4℃
o Refer to assay-specific information above to determine antibody concentration
o Apply to completely cover tissues; let incubate in slide staining tray with lid closed
18h
While slides incubate with overnight primary antibodies:
o Discard protein blocking and leftover antibody dilution reagents
The next day:
o Prepare anti-IBA-1 primary antibody by adding antibody to 1% BSA in PBS. Refer to assay-specific information above to determine the appropriate antibody dilution to use. Also refer to assay controls information for any slides that should not receive antibody. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. Mix diluted antibody well before use.
• Decant slide holder and again place flat in slide staining tray
o Do not wash with PBS-T between decanting overnight antibodies and applying IBA-1 antibody
• Incubate with diluted primary antibody (anti-IBA-1) 1 hour RT
o Refer to assay-specific information above to determine antibody concentration
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 1 hour primary antibody:
o Discard leftover antibody dilution reagents
o Add ~200 mL PBS-T to each of two wash trays
o Prepare cocktail of secondary antibodies by adding antibodies to 1% BSA in PBS. Refer to antibody dilutions/incubations above to determine the appropriate antibody dilution to use. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. Mix diluted antibody well before use.
Secondary Antibody
Secondary Antibody
1h 5m
1h 5m
• Decant slide holder and again place flat in slide staining tray
• Incubate with diluted secondary antibodies 1 hour RT
o Refer to assay-specific information above to determine antibody concentration
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 are incubating with secondary antibody:
o Discard remaining primary antibody reagents
o Add ~200 mL PBS-T to each of two wash trays
Mounting
Mounting
50m
50m
· Remove slides from PBS-T and decant excess liquid
· Apply 2-3 drops of ProLong Gold mounting media to the slide
o Select mounting media with or without DAPI depending on the stain or control required.
o Refer to assay controls information above for more details.
· Overlay with #1.5 coverslip
· Air dry slides RT for 30 min in the dark
· Transfer slides to an opaque slide folder or box and store at 4C
· Image within two weeks
o Refer to Imaging Protocol below for instructions on visualization via spectral demultiplexing on a confocal microscope
50m
While slides are air drying:
· Discard secondary antibody and slide mounting reagents
Protocol references
Contributions/Acknowledgements
· Staining protocol was developed and executed by Dr. Jayne Wiarda
· Imaging protocol was developed and executed by Adrienne Shircliff
· We thank Colin Stoy and Dr. Eraldo Zanella for additional wet lab assistance