TITLE: Dual staining for gamma delta and alpha beta T cell detection in formalin-fixed, paraffin-embedded (FFPE) pig tissues
AUTHORS: Jayne E Wiarda

[DESCRIPTION]
A combined staining protocol for CD3e immunohistochemistry (IHC) and TRDC RNA in-situ hybridization (RNA-ISH) created for in-situ identification of porcine gamma delta T cells (TRDC+) and alpha beta T cells (TRDC-CD3e+)



[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 but not over-fixed as to over-fragment RNA. Tissues no thicker than 0.5 centimeters should be freshly harvested and placed into 10% neutral-buffered formalin (NBF; 3.7% formaldehyde) at a tissue:fixative volume no more than 1:20. Tissues should be fixed in 10% NBF for 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.

[GUIDELINES]
Assay Controls:
Here are a few controls you can use to ensure assay is working correctly:
IHC controls:
            o  Negative control (primary antibody only)
                    §  This slide receives 0.05% PBS-T in place of secondary antibody
            o  Negative control (secondary antibody only)
                    §  This slide receives co-detection antibody diluent in place of diluted primary antibody
RNA-ISH controls:
            o  Positive control
                    §  This slide is incubated with PPIB probe
            o  Negative control
                    §  This slide is incubated with DapB probe

Assay variations:
This assay can be used to detect other proteins or transcripts as well or for detection of protein/RNA in other species by adjusting the antibodies and probes used. Antibodies must be able to detect proteins following heat-induced target retrieval outlined in protocol. 

This assay uses chromogens that cause the RNA signal to dominate the protein signal (i.e. green RNA signal covers up the lighter red protein signal when markers are co-expressed). Other chromogen combinations can be used to instead have the protein signal dominate the RNA signal. For example, Fast Red AP chromogen that comes with the RNAscope kit can be used to detect RNA signal, followed by protein detection with a darker DAB chromogen, such as DAB-Nickel (black), or other DAB chromogens in colors like blue or green.

Parameters for some steps (e.g. antibody incubations, protease incubations, target retrieval) may need to be further optimized for different tissues or targets.

[STEPS]
SECTION: Baking
1. Before baking slides:
Preheat a dry oven to 60℃
Load slides for assay into vertical slide rack

Bake slides 30 min 60℃
Optional stopping point: store slides in a dry place & use within 1 week

While slides bake:
Prepare 0.05% PBS-T (can store at RT up to 1 month)
Prepare 1X Co-Detection Target Retrieval solution by adding 1 bottle (70 mL) Co-Detection Target Retrieval Reagent (10X stock concentration) to 630 mL distilled water (can store at 4℃ up to 1 month)
SECTION: Deparaffinizing
2. Immediately before deparaffinizing:
Add ~200 mL xylenes to each of two clearing agent dishes in a fume hood
Add ~200 mL 100% ethanol to each of two staining dishes in a fume hood

Submerge slide in fresh xylenes 5 min RT
Submerge slide in fresh xylenes 5 min RT
Submerge slides in fresh100% ethanol 5 min RT
Submerge slides in fresh100% ethanol 5 min RT
Air dry slides ~5 min or until completely dry
Optional stopping point: store slides in a dry place & use within 24 hours

While slides deparaffinize:
Prepare decloaking chamber:
---------- Pour 500 mL distilled water into central chamber
---------- Pour 200 mL distilled water into left/right staining dishes
---------- Pour 200 mL prepared Co-Detection Target Retrieval solution into middle staining dish
Turn off dry oven
SECTION: Tissue Quenching
3. Immediately before tissue quenching:
Preheat the prepared decloaking chamber, programmed for 15 min at 95℃
---------- Chamber will take exactly 15 min to preheat, and there will be a 2 min window to add slides before chamber pressurizes & locks

Unload slides from vertical slide rack and place on flat surface of bench top
Incubate with Hydrogen Peroxide 10 min RT
---------- Invert bottle immediately before use; apply drops to completely cover tissues; let incubate on bench top
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

While slides incubate with Hydrogen Peroxide:
Discard deparaffinizing reagents 
Add ~200 mL distilled water to each of two staining dishes

SECTION: Target Retrieval
4. Leave slides in water at RT until decloaker is preheated (<5 min)
Once decloaker has preheated, submerge slide rack in preheated distilled water 10 sec (left or right dishes in decloaker)
Submerge slide rack in preheated 1X Co-detection Target Retrieval solution 15 min 95℃
---------- Once slides are places in center staining dish of decloaker, close the decloaker (make sure pressure valve is in place to hold pressure when replacing lid) & wait for alarm to go off in 15 min
Release decloaker chamber pressure valve & open chamber
Submerge slide rack in preheated distilled water 10 sec (left or right dishes in decloaker)
Submerge slide rack in fresh distilled water, dunking 3-5 times
Submerge slide rack infresh distilled water, dunking 3-5 times
Submerge slide rack infresh PBS-T, dunking 3-5 times
Leave slides in PBS-T

While slides incubate in 1X target retrieval solution:
Discard tissue quenching reagents 
Add ~200 mL distilled water to each of two staining dishes
Add ~200 mL PBS-T to one staining dish
Prepare humidified slide staining tray by adding water to bottom & placing lid on top
Add CD3e antibody to Co-Detection Antibody Diluent at a 1:50 dilution. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting.
SECTION: Hydrophobic Barrier
5. Apply hydrophobic barrier around each tissue
---------- 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 slide flat in 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)
SECTION: Primary Antibody
6. Decant slides and again place flat in slide staining tray
Incubate with diluted primary antibody 60 min RT; place lid back on slide staining tray during this time
Remove slides from slide staining tray, decant, and transfer to vertical slide rack
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

While slides are incubating with primary antibody:
Discard target retrieval reagents 
Add ~200 mL PBS-T to each of three staining dishes
Add ~200 mL 10% NBF to one staining dish in a fume hood
SECTION: Antibody Cross-linking
7. Submerge slide rack in 10% NBF 30 min RT
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

While slides are incubating with 10% NBF:
Discard primary antibody reagents 
Add ~200 mL PBS-T to each of three staining dishes
Prepare HybEZ Oven:
---------- Place humidifying paper within the humidity control tray & apply distilled water to fully wet paper
---------- Place humidifying tray into HybEZ oven and clamp down the gasket to seal
---------- Preheat oven to 40℃ for at least 30 minutes before use
SECTION: Protease
8. Transfer slides into EZ-Batch Slide Holder, taking care not to let tissues dry out
Incubate with Protease Plus 15 min 40℃
---------- Invert bottle immediately before use; apply drops to completely cover tissues & transfer slide holder to humidifying tray within HybEZ oven
Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder)
Submerge slide holder in fresh distilled water, dunking 3-5 times
Submerge slide holder in fresh distilled water, dunking 3-5 times

While slides are incubating with protease:
Empty the slide staining tray used for primary antibody incubations & put away
Discard antibody cross-linking reagents 
Add ~200 mL distilled water to each of two wash trays
Preheat RNAscope probes to 40℃ for 10 min before use; this can be done by placing them inside the HybEZ oven during protease incubation
SECTION: Probe Hybridization
9. Decant slides (without removing slides from holder)
Incubate with appropriate RNAscope probe 2 hours 40℃
---------- Invert bottle immediately before use; apply drops to completely cover tissues & transfer slide holder to humidifying tray within HybEZ oven
Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder)
Submerge slide holder in fresh 1X wash buffer 2 min RT
Submerge slide holder in fresh 1X wash buffer 2 min RT

While slides are incubating with probes:
Discard protease reagents 
Prepare 1X wash buffer by adding 1 bottle (60 mL) Wash Buffer (10X stock concentration) to 3.94 L distilled water
---------- If 10X Wash Buffer solution has a precipitant formed, preheat bottle at 37℃ for 1 hour before adding to distilled water
---------- Will have to prepare another batch of 1X wash buffer later in protocol, after first batch runs out. Alternatively, prepare both batches at once (120 mL 1X Wash Buffer + 7.88 L distilled water)
---------- Store at RT up to one month
Add ~200 mL 1X wash buffer to each of two wash trays
Place AMPs from RNAscope detection kit at RT for at least 30 min before use (should be RT when used)
SECTION: RNA Signal Amplification
10. Decant slides (without removing slides from holder)
Incubate with AMP1 30 min 40℃
---------- Invert bottle immediately before use; apply drops to completely cover tissues & transfer slide holder to humidifying tray within HybEZ oven
Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder)
Submerge slide holder in fresh 1X wash buffer 2 min RT
Submerge slide holder in fresh 1X wash buffer 2 min RT
Decant slides (without removing slides from holder)
Incubate with AMP2 15 min 40℃
---------- Invert bottle immediately before use; apply drops to completely cover tissues & transfer slide holder to humidifying tray within HybEZ oven
Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder)
Submerge slide holder in fresh 1X wash buffer 2 min RT
Submerge slide holder in fresh 1X wash buffer 2 min RT
Decant slides (without removing slides from holder)
Incubate with AMP3 30 min 40℃
---------- Invert bottle immediately before use; apply drops to completely cover tissues & transfer slide holder to humidifying tray within HybEZ oven
Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder)
Submerge slide holder in fresh 1X wash buffer 2 min RT
Submerge slide holder in fresh 1X wash buffer 2 min RT
Decant slides (without removing slides from holder)
Incubate with AMP4 15 min 40℃
---------- Invert bottle immediately before use; apply drops to completely cover tissues & transfer slide holder to humidifying tray within HybEZ oven
Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder)
Submerge slide holder in fresh 1X wash buffer 2 min RT
Submerge slide holder in fresh 1X wash buffer 2 min RT
Decant slides (without removing slides from holder)
Incubate with AMP5 30 min RT
---------- Invert bottle immediately before use; apply drops to completely cover tissues & transfer slide holder to humidifying tray left on bench top
Remove slide holder from humidifying tray & decant (without removing slides from holder)
Submerge slide holder in fresh 1X wash buffer 2 min RT
Submerge slide holder in fresh 1X wash buffer 2 min RT
Decant slides (without removing slides from holder)
Incubate with AMP6 15 min RT
---------- Invert bottle immediately before use; apply drops to completely cover tissues & transfer slide holder to humidifying tray left on bench top
Remove slide holder from humidifying tray & decant (without removing slides from holder)
Submerge slide holder in fresh 1X wash buffer 2 min RT
Submerge slide holder in fresh 1X wash buffer 2 min RT

During each AMP incubation:
Discard reagents from previous incubation step 
Add ~200 mL 1X wash buffer to each of two wash trays
SECTION: RNA Detection
11. Immediately before RNA detection:
Prepare diluted green AP chromogen by adding 80 uL HIGHDEF Green AP Chromogen per 920 uL HIGHDEF Green AP Substrate. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. Store in the dark due to light sensitivity

Decant slides (without removing slides from holder)
Incubate with diluted green AP chromogen 20 min RT
---------- Pipette well to mix immediately before use; pipette appropriate volumes to completely cover tissues & transfer slide holder to humidifying tray left on bench top
Remove slide holder from humidifying tray & decant (without removing slides from holder)
Submerge slide holder in fresh1X wash buffer 2 min RT
Submerge slide holder in fresh 1X wash buffer 2 min RT

While slides are incubating with green AP chromogen:
Discard remaining AMP detection reagents 
Add ~200 mL 1X wash buffer to each of two wash trays
SECTION: Tissue Blocking
12. Decant slides (without removing slides from holder)
Incubate with Co-Detection Blocker 15 min 40℃
---------- Invert bottle immediately before use; apply drops to completely cover tissues & transfer slide holder to humidifying tray left on bench top
Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder)
Submerge slide holder in fresh 1X wash buffer 2 min RT
Submerge slide holder in fresh 1X wash buffer 2 min RT

While slides are incubating with Co-Detection Blocker:
Discard RNA detection reagents 
Add ~200 mL 1X wash buffer to each of two wash trays
Add ~200 mL PBS-T to one wash tray
SECTION: Secondary Antibody
13. Decant slides (without removing slides from holder)
Incubate with anti-rabbit HRP polymer 30 min RT
---------- Invert bottle immediately before use; apply drops to completely cover tissues & transfer slide holder to humidifying tray on bench top
Remove slide holder from humidifying tray & decant (without removing slides from holder)
Submerge slide holder in fresh PBS-T 2 min RT
Submerge slide holder in fresh PBS-T 2 min RT

While slides are incubating with secondary antibody:
Discard tissue blocking reagents 
Add ~200 mL 1X wash buffer to each of two wash trays
Add ~200 mL PBS-T to one wash tray (if you only have two wash trays, complete this step after one PBS-T incubation after secondary antibody incubation)
Turn off HybEZ oven
SECTION: Protein Detection
14. Immediately before protein detection:
Prepare diluted red horseradish peroxidase (HRP) chromogen by adding 1 drop PermaRed/HRP Chromogen per 1 mL PermaRed/HRP Substrate Buffer. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. Store in the dark due to light sensitivity

Decant slides (without removing slides from holder)
Incubate with diluted red HRP chromogen 10 min RT
---------- Pipette well to mix immediately before use; pipette appropriate volumes to completely cover tissues & transfer slide holder to humidifying tray left on bench top
---------- At ~3 and ~6 min into incubation, use a pipette to redistribute chromogen on each slide (this chromogen tends to sediment and settle on slides, causing uneven staining if it is not redistributed)
Remove slide holder from humidifying tray, decant, & transfer slides into 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
Submerge slide rack in fresh distilled water, dunking 3-5 times

While slides are incubating with red HRP chromogen:
Discard secondary antibody reagents 
Add ~200 mL distilled water to each of six staining dishes
Add ~200 mL hematoxylin to one staining dish
SECTION: Counterstaining
15. Submerge slide rack in hematoxylin 2 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
SECTION: Mounting
16. Decant slides (without removing slides from rack)
Air dry slides ~20 min or until completely dry
Mount slides by adding 2-4 drops of 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; stores slides in the dark at RT

While slides are air drying:
Discard protein detection and counterstaining reagents