TITLE: Tri-plex staining for IBA-1, CD4, and CSF1R detection in formalin-fixed, paraffin-embedded (FFPE) pig tissues
AUTHORS: Jayne E Wiarda, Crystal L Loving

[DESCRIPTION]
A protocol for staining of protein (IBA-1) and RNA (CD4, CSF1R) in pig tissues

[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) or 4% paraformaldehyde (PFA) at a ratio of at least 20 volumes fixative per one volume tissue. Tissues should be fixed 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.

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

Baking
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 beforen 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

Deparaffinizing
Submerge slide in fresh xylenes 5 min RT
Submerge slide in fresh xylenes 5 min RT
Submerge slides in fresh 100% ethanol 5 min RT
Submerge slides in fresh 100% 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:
o  ----------Pour 500 mL distilled water into central chamber
o  ----------Pour 200 mL distilled water into left/right staining dishes
o  ----------Pour 200 mL prepared Co-Detection Target Retrieval solution into middle staining dish
Turn off dry oven
Prepare HybEZ Oven:
o  ----------Place humidifying paper within the humidity control tray & apply distilled water to fully wet paper
o  ----------Place humidifying tray into HybEZ oven and clamp down the gasket to seal
o  ----------Preheat oven to 40℃ for at least 30 minutes before use
SECTION: Tissue Quenching
3. Immediately before tissue quenching:
Preheat the prepared decloaking chamber, programmed for 15 min at 95℃
o  ----------Chamber will take exactly 15 min to preheat, and there will be a 2 min window to add slides before chamber pressurizes & locks

Tissue Quenching
Unload slides from vertical slide rack and place on flat surface of bench top
Incubate with Hydrogen Peroxide 10 min RT
o  ----------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. Target Retrieval
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℃
o  ----------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 in fresh distilled water, dunking 3-5 times
Submerge slide rack in fresh 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 IBA-1 antibody to Co-Detection Antibody Diluent at a 1:1000 dilution (0.5 ug/mL). Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting.
SECTION: Hydrophobic Barrier
5. Hydrophobic Barrier
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 ImmEdge 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. Primary Antibody
Decant slides and again place flat in slide staining tray
Incubate with diluted primary antibody RT 1 hour; 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. Antibody cross-linking
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:
o  ----------Place humidifying paper within the humidity control tray & apply distilled water to fully wet paper
o  ----------Place humidifying tray into HybEZ oven and clamp down the gasket to seal
o  ----------Preheat oven to 40℃ for at least 30 minutes before use
SECTION: Protease
8. Protease
Transfer slides into EZ-Batch Slide Holder, taking care not to let tissues dry out
Incubate with Protease Plus 15 min 40℃
o  ----------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. Once preheated, add CD4-C2 probe to CSF1R-C1 probe at a dilution of 1:50. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. 

SECTION: Probe Hybridization
9. Probe Hybridization
Decant slides (without removing slides from holder)
Incubate with appropriate RNAscope probe cocktail 2 hours 40℃
o  ----------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 2.94L distilled water
o  ----------If 10X Wash Buffer solution has a precipitant formed, preheat bottle at 37℃ for 1 hour before adding to distilled water
o  ----------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 + 5.88 L distilled water)
o  ----------Store at RT up to one month
Add ~200 mL 1X wash buffer to each of two wash trays
If performing optional stopping point (next step):
o  ----------Prepare 5X SSC buffer by combining 60 mL 20X SSC with 180 mL distilled water
o  ----------Add 240 mL 5X SSC buffer to staining dish
If omitting optional stopping point (next step):
o  ----------Place AMPs from RNAscope Multiplex Fluorescent Detection kit at RT for at least 30 min before use (should be RT when used)
SECTION: Stopping Point (optional)
10. Stopping Point (optional)
Transfer slides from slide holder into slide rack
Submerge slide rack in fresh 5X SSC buffer overnight at RT with lid
Decant slides and transfer into slide holder
Submerge slide holder in fresh 1X wash buffer 2 min RT
Submerge slide holder in fresh 1X wash buffer 2 min RT

The next day:
Place AMPs from RNAscope Multiplex Fluorescent Detection kit at RT for at least 30 min before use (should be RT when used)
Add ~200 mL 1X wash buffer to each of two wash trays
SECTION: RNA Detection
11. RNA Detection
Incubate with AMP1 30 min 40℃
o  ----------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 30 min 40℃
o  ----------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 15 min 40℃
o  ----------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 HRP-C1 15 min 40℃
o  ----------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 diluted Opal 690 30 min 40℃
o  ----------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 fresh 1X wash buffer 2 min RT
Submerge slide holder in fresh 1X wash buffer 2 min RT
Incubate with HRP blocker 15 min 40℃
o   ----------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
Decant slides (without removing slides from holder)
Incubate with HRP-C2 15 min 40℃
o  ----------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 diluted Opal 570 30 min 40℃
o  ----------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 fresh 1X wash buffer 2 min RT
Submerge slide holder in fresh 1X wash buffer 2 min RT
Incubate with HRP blocker 15 min 40℃
o   ----------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

During each incubation:
Discard reagents from previous incubation step 
Add ~200 mL 1X wash buffer to each of two wash trays

Immediately before Opal690 incubation:
Prepare diluted Opal fluorophore by diluting Opal690 into Multiplex TSA Buffer at 1:1200 dilution. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. Store in the dark due to light sensitivity.

Immediately before Opal570 incubation:
Prepare diluted Opal fluorophore by diluting Opal570 into Multiplex TSA Buffer at 1:750 dilution. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. Store in the dark due to light sensitivity.

While slides are incubating with last HRP blocker:
Add ~200 mL 1X wash buffer to each of two wash trays
Add ~200 mL PBS-T to one wash tray
SECTION: Protein Detection
12. Immediately before protein detection:
Prepare diluted secondary antibody by diluting Alexa Fluor 488 F(ab’) fragment of goat anti-rabbit IgG into Co-detection Antibody Diluent at a 1:1000 mdilution. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting. Store in the dark due to light sensitivity.

Protein Detection
Decant slides (without removing slides from holder)
Incubate with diluted secondary antibody 1 hour RT
o  ----------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: Nuclei Staining and Coverslipping
13. Nuclei Staining and Coverslipping
One at a time, remove slides from slide holder and:
o   ----------Apply DAPI 30 sec RT
o   ----------Decant slide to remove DAPI
o   ----------Mount slides by adding 2-4 drops of ProLong Gold antifade 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 30 min
Store at 4℃ and image within two weeks

While slides are air drying:
Discard protein detection reagents