TITLE: In situ Ki-67 detection in formalin-fixed, paraffin-embedded (FFPE) pig tissues
AUTHORS: Jayne E Wiarda, Crystal Loving

[DESCRIPTION]
An immunohistochemistry (IHC) staining protocol for in situ identification of porcine Ki-67

[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.

[GUIDELINES]
Assay Controls:
Here are a few controls you can use to ensure assay is working correctly:
IHC controls:
oNegative control (primary antibody only)
§This slide receives 0.05% PBS-T in place of secondary antibody
oNegative control (secondary antibody only)
§This slide receives co-detection antibody diluent in place of diluted primary antibody
oBatch control 
§If performing staining across multiple batches, include serial sections of one tissue in each batch

Assay variations:
Parameters for some steps (e.g. antibody incubations, antigen retrieval, chromogen incubations, counterstaining) may need to be further optimized for different tissues or targets.

[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 20 min 60℃

While slides bake:
Prepare 0.05% PBS-T (can store at RT up to 1 month)
SECTION: Deparaffinizing &amp; Rehydrating
2. Immediately before deparaffinizing:
Add ~200 mL xylenes to each of three clearing agent dishes in a fume hood
Add ~200 mL 100% ethanol to each of two staining dishes in a fume hood
Add ~200 mL 95% ethanol to a staining dish in a fume hood
Add ~200 mL 85% ethanol to a staining dish in a fume hood
Add ~200 mL 70% ethanol to a staining dish in a fume hood
Add ~200 mL distilled water to a staining dish in a fume hood
Add ~200 mL PBS-T to a staining dish in a fume hood

Deparaffinizing & Rehydrating
Submerge slide in fresh xylenes 5 min RT
Submerge slide in fresh xylenes 5 min RT
Submerge slide in fresh xylenes 5 min RT
Submerge slides in fresh 100% ethanol 1 min RT
Submerge slides in fresh 100% ethanol 1 min RT
Submerge slides in fresh 95% ethanol 1 min RT
Submerge slides in fresh 85% ethanol 1 min RT
Submerge slides in fresh 70% ethanol 1 min RT
Submerge slides in fresh distilled water 3 min RT
Submerge slides in fresh PBS-T for transport

While slides deparaffinize/rehydrate:
Turn off dry oven
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)
Prepare the steamer
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
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
SECTION: Heat-Induced Epitope Retrieval
3. Heat-Induced Epitope Retrieval
Leave slides in PBS-T 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), click “Skip” on screen since slides are loaded, & 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 infresh PBS-T
Leave slides in PBS-T

While slides incubate in 1X co-detection target retrieval solution:
Discard deparaffinizing & rehydrating reagents 
Add ~200 mL PBS-T to one staining dish
Prepare humidified slide staining tray by adding water to bottom & placing lid on top
SECTION: Hydrophobic Barrier
4. Hydrophobic Barrier
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)
Leave slides in slide staining tray
SECTION: Tissue Quenching
5. Tissue Quenching 
Decant slides and again place flat in slide staining tray
Incubate with Dual Endogenous Enzyme Block 10 min RT
---------- Invert bottle immediately before use; apply drops 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 PBS-T 2 min RT
Submerge slide rack in fresh PBS-T 2 min RT

While slides incubate with enzyme block:
Discard epitope retrieval reagents 
Add ~200 mL PBS-T to each of two staining dishes
SECTION: Protein Blocking
6. Protein Blocking
Decant slides and again place flat in slide staining tray
Incubate with Protein Block 20 min RT
oInvert bottle immediately before use; apply drops 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 PBS-T 2 min RT
Submerge slide rack in fresh PBS-T 2 min RT

While slides incubate with protein block:
Discard tissue quenching reagents 
Prepare primary antibody by adding anti-Ki-67 antibody to Co-Detection Antibody Diluent at a dilution of 2.5 ug/mL (1:100 dilution if stock antibody concentration is 250 ug/mL). Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting.
SECTION: Primary Antibody
7. Primary Antibody
Decant slides and again place flat in slide staining tray
Incubate with diluted primary antibody overnight at 4℃
---------- Apply drops to completely cover tissues; let incubate in slide staining tray with lid closed
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

While slides are incubating with primary antibody:
Discard protein blocking reagents 
SECTION: Secondary Antibody
8. The next day:
Add ~200 mL PBS-T to each of two staining dishes

Secondary Antibody
Decant slides and again place flat in slide staining tray
Incubate with anti-mouse HRP polymer 30 min RT
---------- Invert bottle immediately before use; apply drops 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 PBS-T 2 min RT
Submerge slide rack in fresh PBS-T 2 min RT

While slides are incubating with secondary antibody:
Discard remaining primary antibody reagents 
Add ~200 mL PBS-T to each of two staining dishes
SECTION: Chromogen Detection
9. Immediately before chromogen detection:
Prepare diluted DAB chromogen by adding 1 drop DAB substrate per 1 mL substrate buffer. Total volume to use is dependent on tissue sizes. Make sure to mix reagents thoroughly. Store in the dark due to light sensitivity

Chromogen Detection
Decant slides and again place flat in slide staining tray
Incubate with diluted DAB chromogen 7 min RT
---------- Pipette well to mix immediately before use; pipette appropriate volumes 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 PBS-T 2 min RT
Submerge slide rack in fresh PBS-T 2 min RT

While slides are incubating with DAB chromogen:
Discard secondary antibody reagents 
Add ~200 mL PBS-T to each of two staining dishes
Add ~200 mL 25% hematoxylin to one staining dish
---------- Prepare by combining 150 mL distilled water with 50 mL Gill’s Hematoxylin
Add ~200 mL distilled water to each of three staining dishes
Add ~200 mL 95% ethanol to a staining dish in a fume hood
Add ~200 mL 100% ethanol to each of three staining dishes in a fume hood
Add ~200 mL Pro-Par to each of three clearing agent dishes in a fume hood
SECTION: Counterstaining
10. Counterstaining
Submerge slide rack in diluted hematoxylin 15 sec 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
11. Mounting
Submerge slides in fresh 95% ethanol 1 min RT
Submerge slides in fresh 100% ethanol 1 min RT
Submerge slides in fresh 100% ethanol 1 min RT
Submerge slides in fresh 100% ethanol 1 min RT
Submerge slides in fresh Pro-Par 5 min RT
Submerge slides in fresh Pro-Par 5 min RT
Submerge slides in fresh Pro-Par 5 min RT
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

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