TITLE: In situ immunoglobulin M (IgM) 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 IgM in pig tissue. 

[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) 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:
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 1% BSA in PBS in place of diluted primary antibody
o  Batch control 
§ If performing staining across multiple batches, include serial sections of one tissue in each batch that has positive staining for Salmonella

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 humidified slide staining tray by adding water to bottom & placing lid on top
Add ~200 mL PBS-T to each of two staining dishes
SECTION: Hydrophobic Barrier
3. 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
SECTION: Protease Digestion
4. 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
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
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
SECTION: Protein Blocking
6. 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 IgM antibody to 1% BSA in PBS at a dilution of 0.05 uL/mL (1:20,000 dilution). 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℃
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
SECTION: Secondary Antibody
8. 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
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 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
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