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)

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 humidified slide staining tray by adding water to bottom & placing lid on top

• Add ~200 mL PBS-T to each of two staining dishes

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 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

Protease Digestion

• Decant slides and again place flat in slide staining tray

• Incubate with Proteinase K 3 min RT o ----------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

Tissue Quenching

• Decant slides and again place flat in slide staining tray

• Incubate with Dual Endogenous Enzyme Block 10 min RT o ----------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 deparaffinizing & rehydrating and protease digestion reagents
• Add ~200 mL PBS-T to each of two staining dishes

Protein Blocking

• Decant slides and again place flat in slide staining tray

• Incubate with Protein Block 20 min RT o ----------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 protein block:

• Discard tissue quenching reagents
• Prepare primary antibody by adding IgG antibody to 1% BSA in PBS at a dilution of 0.02 uL/mL (1:50,000 dilution). Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting.

Primary Antibody

• Decant slides and again place flat in slide staining tray
• Incubate with diluted primary antibody overnight at 4℃ o ----------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

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-rabbit HRP polymer 30 min RT o ----------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

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 2 min RT o ----------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 o ----------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

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

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