TITLE: Tri-plex staining for PAX5, CD4, and CSF1R detection in formalin-fixed, paraffin-embedded (FFPE) pig tissues
AUTHORS: Jayne E Wiarda, Alyssa Ivy, Hannah Mazon, Colin Stoy, sage.becker, Crystal Loving

[DESCRIPTION]
A protocol for staining of protein (PAX5) and RNA (CD4, CSF1R) in pig tissues.

[BEFORE_START]
• Preheat a dry oven to 60℃.
• Trim tissue on slides to smaller size if needed. 
• Load slides for assay into vertical slide rack

[GUIDELINES]
Supporting Information

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) 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.

Assay Controls:

Here are a few controls you can use to ensure the assay is working correctly: 
• IHC controls: 

     o Negative control (primary antibody only)
 This slide receives co-detection antibody diluent in place of diluted secondary antibody. 
     o Negative control (secondary antibody only)
 This slide receives co-detection antibody diluent 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 PAX5. 

• 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:

Parameters for some steps (e.g. antigen retrieval, antibody incubations, protease plus incubation, fluorophore/opal incubations, some AMP incubations, counterstaining) may need to be further optimized for different tissues or targets. This protocol is likely applicable to tissues of other species. Consult ACD for optimization assistance. 

[STEPS]
SECTION: Baking
1. Bake slides 30 min 60 °C.

 

SECTION: Baking
2. While slides bake:
SECTION: Baking
3. Immediately before deparaffinizing:
SECTION: Deparaffinizing
4. Submerge slides in fresh xylenes 5 min Room temperature.

 

SECTION: Deparaffinizing
9. While slides deparaffinize:



 

SECTION: Tissue Quenching
10. Unload slides from vertical slide rack and place on flat surface of bench top.
SECTION: Tissue Quenching
15. While slides incubate with Hydrogen Peroxide:
SECTION: Target Retrieval
16. Leave slides in water at Room temperature until decloaker is preheated (<5 min).
SECTION: Target Retrieval
24. While slides incubate in 1X target retrieval solution:

SECTION: Hydrophobic Barrier
25. Apply hydrophobic barrier around each tissue.

  
SECTION: Primary Antibody
30. Decant slides and again place flat in slide staining tray.
SECTION: Primary Antibody
32. While slides are incubating with primary antibody: 
SECTION: Primary Antibody
33. The next day: 
SECTION: Primary Antibody
34. While slides are sitting in PBS-T:
SECTION: Antibody cross-linking
35. Submerge slide rack in 10% NBF 30 min Room temperature.
SECTION: Antibody cross-linking
39. While slides are incubating with 10% NBF:

Add ~200 mL PBS-T to each of three staining dishes.
SECTION: Protease
41. Transfer slides into EZ-Batch Slide Holder, taking care not to let tissues dry out.
SECTION: Protease
46. While slides are incubating with protease:
SECTION: Probe Hybridization
47. Decant slides (without removing slides from holder).
SECTION: Probe Hybridization
52. While slides are incubating with probes:
SECTION: RNA Detection
57. Decant slides (without removing slides from holder). 
SECTION: RNA Detection
59. Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder).

SECTION: RNA Detection
64. Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder).
SECTION: RNA Detection
69. Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder).
SECTION: RNA Detection
74. Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder).
SECTION: RNA Detection
75. Immediately before TSA Vivid Fluorophore 520 incubation:

Prepare diluted fluorophore by diluting TSA Vivid Fluorophore 520 into Multiplex TSA Buffer at a dilution of 1:1000 at Room temperature. Total volume to use is dependent on tissue sizes. 

 

SECTION: RNA Detection
76. Decant slides (without removing slides from holder).
SECTION: RNA Detection
78. Remove slide holder from humidifying tray & decant (without removing slides from holder).
SECTION: RNA Detection
82. Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder).
SECTION: RNA Detection
87. Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder).
SECTION: RNA Detection
90. Immediately before Opal 650 incubation:

Prepare diluted Opal fluorophore by diluting Opal 650 into Multiplex TSA Buffer at a dilution of 1:1000 at RT. Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting.

 

SECTION: RNA Detection
91. Decant slides (without removing slides from holder).
SECTION: RNA Detection
93. Remove slide holder from humidifying tray & decant (without removing slides from holder).
SECTION: RNA Detection
97. Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder).
SECTION: RNA Detection
100. During each incubation: 
SECTION: Protein Blocking
101. Decant slides (without removing slides from holder). 
SECTION: Protein Blocking
103. Remove slide holder from humidifying tray & decant (without removing slides from holder).
SECTION: Protein Blocking
106. While slides are incubating with protein block:
SECTION: Protein Blocking
107. Immediately before protein detection:

Prepare diluted secondary antibody by adding Alexa FluorTM 594 donkey anti-rat IgG to Co-Detection Antibody Diluent at a dilution of 1:500 at Room temperature. Total volume to use is dependent on tissue sizes. 

 

SECTION: Protein Detection
108. Decant slides (without removing slides from holder).
SECTION: Protein Detection
113. While slides are incubating with secondary antibody:
SECTION: Nuclei Staining and Cover Slipping
114. One at a time, remove slides from slide holder and:
SECTION: Baking
2.1. Prepare 0.05% PBS-T (can store at Room temperature up to 1 month).
SECTION: Baking
2.2. Prepare 1X Co-Detection Target Retrieval solution by adding 1 bottle (70 mL) of Co-Detection Target Retrieval Reagent (10X stock concentration) to 630 mL distilled water (can store at 4 °C up to 1 month).
SECTION: Baking
3.1. Add ~200 mL xylenes to each of two clearing agent dishes in a fume hood.
SECTION: Baking
3.2. Add ~200 mL 100% ethanol to each of two staining dishes in a fume hood.
SECTION: Deparaffinizing
5. Submerge slides in fresh xylenes 5 min Room temperature.
SECTION: Deparaffinizing
6. Submerge slides in fresh 100% ethanol 5 min Room temperature .
SECTION: Deparaffinizing
7. Submerge slides in fresh 100% ethanol 5 min Room temperature.
SECTION: Deparaffinizing
8. Air dry slides ~5 min or until completely dry.

SECTION: Deparaffinizing
9.1. • Prepare decloaking chamber:

o Pour 500 mL distilled water into central chamber.
o Pour 200 mL distilled water into each left and right staining dishes.
o Pour 200 mL prepared 1X Co-Detection Target Retrieval solution into middle staining dish.

 

SECTION: Deparaffinizing
9.2. Immediately before tissue quenching: 


 

Chamber will take exactly 15 min to preheat, and there will be a 2 min window to add slides before chamber pressurizes & locks.

SECTION: Tissue Quenching
11. Incubate with Hydrogen Peroxide 10 min Room temperature.

 


SECTION: Tissue Quenching
12. Decant slides and transfer to vertical slide rack.
SECTION: Tissue Quenching
13. Submerge slide rack in fresh distilled water, dunking 3-5 times.
SECTION: Tissue Quenching
14. Submerge slide rack in fresh distilled water, dunking 3-5 times.
SECTION: Tissue Quenching
15.1. Discard deparaffinizing reagents.
SECTION: Tissue Quenching
15.2. Add ~200 mL distilled water to each of two staining dishes.
SECTION: Target Retrieval
17. Once decloaker has preheated, submerge slide rack in preheated distilled water 10 s (left dish in decloaker).
SECTION: Target Retrieval
18. Submerge slide rack in preheated 1X Co-Detection Target Retrieval solution15 min 95 °C.

   o Once slides are placed in center staining dish of decloaker, close the decloaker  & wait for alarm to go off in 15 min. 

 

SECTION: Target Retrieval
19. Release decloaker chamber pressure valve & open chamber.
SECTION: Target Retrieval
20. Submerge slide rack in fresh distilled water, dunking 3-5 times.
SECTION: Target Retrieval
21. Submerge slide rack in fresh distilled water, dunking 3-5 times.
SECTION: Target Retrieval
22. Submerge slide rack in fresh PBS-T, dunking 3-5 times.
SECTION: Target Retrieval
23. Leave slides in PBS-T.
SECTION: Target Retrieval
19.1. Submerge slide rack in preheated distilled water 10 s (right dish in decloaker).
SECTION: Target Retrieval
24.1. Discard tissue quenching reagents.
SECTION: Target Retrieval
24.2. Add ~200 mL distilled water to each of two staining dishes.
SECTION: Target Retrieval
24.3. Add ~200 mL PBS-T to one staining dish.
SECTION: Target Retrieval
24.4. Prepare humidified slide staining tray by adding water to bottom & placing lid on top.
SECTION: Target Retrieval
24.5. Prepare diluted primary antibody by adding anti-PAX5 antibody to Co-Detection Antibody Diluent at a 1:100 dilution (if stock antibody concentration is 500 1527 ) at Room temperature. Total volume to use is dependent on tissue sizes. 

 

SECTION: Hydrophobic Barrier
26. One by one, unload slides from vertical rack submerged in PBS-T.
SECTION: Hydrophobic Barrier
27. Dry off only the area around the tissue where a barrier will be drawn with ImmEdge Hydrophobic Barrier Pen. 

 

SECTION: Hydrophobic Barrier
28. Draw barrier and place slide flat in the slide staining tray. 
SECTION: Hydrophobic Barrier
29. Using a pipette, apply a small amount of PBS-T within the barrier.

 

SECTION: Primary Antibody
31. Incubate with diluted primary antibody 10 s at 4 °C. 

 

SECTION: Primary Antibody
32.1. Discard target retrieval and primary antibody reagents.
SECTION: Primary Antibody
33.1. Remove slides from slide staining tray, decant, and transfer to vertical slide rack.
SECTION: Primary Antibody
33.2. Submerge slide rack in fresh PBS-T 2 min Room temperature.
SECTION: Primary Antibody
33.3. Submerge slide rack in fresh PBS-T 2 min Room temperature.
SECTION: Primary Antibody
33.4. Submerge slide rack in fresh PBS-T 2 min Room temperature.
SECTION: Primary Antibody
34.1. Add ~200 mL PBS-T to each of three staining dishes.
SECTION: Primary Antibody
34.2. Add ~200 mL 10% NBF to one staining dish in a fume hood.
SECTION: Antibody cross-linking
36. Submerge slide rack in fresh PBS-T 2 min Room temperature.
SECTION: Antibody cross-linking
37. Submerge slide rack in fresh PBS-T 2 min Room temperature.
SECTION: Antibody cross-linking
38. Submerge slide rack in fresh PBS-T 2 min Room temperature.
SECTION: Antibody cross-linking
40. Prepare HybEZ Oven:
SECTION: Antibody cross-linking
40.1. Place humidifying paper within the humidity control tray & apply distilled water to fully wet paper.
SECTION: Antibody cross-linking
40.2. Place humidifying tray into HybEZ oven and clamp down the gasket to seal.
SECTION: Antibody cross-linking
40.3. Preheat oven to 40 °C for at least 30 min before use.
SECTION: Protease
42. Incubate with Protease Plus 15 min 40 °C.

 

SECTION: Protease
43. Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder).
SECTION: Protease
44. Submerge slide holder in fresh distilled water, dunking 3-5 times.
SECTION: Protease
45. Submerge slide holder in fresh distilled water, dunking 3-5 times.
SECTION: Protease
46.1. Empty the slide staining tray used for primary antibody incubation & put away.
SECTION: Protease
46.2. Discard antibody cross-linking reagents.
SECTION: Protease
46.3. Add ~200 mL distilled water to each of two wash trays.
SECTION: Protease
46.4. Preheat RNAscope probes to 40 °C for 10 min before use. This can be done by placing them inside the HybEZ oven during protease incubation. 
SECTION: Protease
46.5. 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. 

 

SECTION: Probe Hybridization
48. Incubate with appropriate RNAscope probe cocktail 120 min 40 °C.

 

SECTION: Probe Hybridization
49. Remove slide holder from HybEZ oven/humidifying tray & decant (without removing slides from holder).
SECTION: Probe Hybridization
50. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: Probe Hybridization
51. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: Probe Hybridization
52.1. Discard protease reagents.
SECTION: Probe Hybridization
52.2. Prepare 1X wash buffer by adding 1 bottle (60 mL) Wash Buffer (10X stock concentration) to 2.94 L distilled water.
SECTION: Probe Hybridization
53. If 10X Wash Buffer solution has a precipitant formed, preheat bottle at 37 °C for 60 min before adding to distilled water.
SECTION: Probe Hybridization
54. Will have to prepare another batch of 1X wash buffer later in protocol, after first batch runs out. 
SECTION: Probe Hybridization
55. Alternatively, prepare both batches at once (120 mL 1X Wash Buffer + 5.88 L distilled water).

 

SECTION: Probe Hybridization
56. Add ~200 mL 1X wash buffer to each of two wash trays.
Place AMPs from RNAscope Multiplex Fluorescent Detection kit at Room temperature for at least 30 min before use (should be Room temperature when used).
SECTION: RNA Detection
58. Incubate with AMP1 30 min 40 °C.

 

SECTION: RNA Detection
60. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature
SECTION: RNA Detection
61. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
62. Decant slides (without removing slides from holder).
SECTION: RNA Detection
63. Incubate with AMP2 30 min 40 °C.

 

SECTION: RNA Detection
65. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
66. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
67. Decant slides (without removing slides from holder).
SECTION: RNA Detection
68. Incubate with AMP3 15 min 40 °C.

 

SECTION: RNA Detection
70. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
71. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
72. Decant slides (without removing slides from holder).
SECTION: RNA Detection
73. Incubate with HRP-C1 15 min 40 °C.

 

SECTION: RNA Detection
74.1. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.

SECTION: RNA Detection
74.2. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature
SECTION: RNA Detection
77. Incubate with diluted TSA Vivid Fluorophore 520 30 min 40 °C.

 

SECTION: RNA Detection
79. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
80. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
81. Incubate with HRP blocker 15 min 40 °C.

 

SECTION: RNA Detection
83. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
84. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
85. Decant slides (without removing slides from holder).
SECTION: RNA Detection
86. Incubate with HRP-C2 15 min 40 °C.

 

SECTION: RNA Detection
88. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
89. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature
SECTION: RNA Detection
92. Incubate with diluted Opal 650 30 min 40 °C.

 

SECTION: RNA Detection
94. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
95. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
96.  Incubate with HRP blocker 15 min 40 °C.

 

SECTION: RNA Detection
98. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
99. Submerge slide holder in fresh 1X wash buffer 2 min Room temperature.
SECTION: RNA Detection
100.1. Discard reagents from previous incubation step.
SECTION: RNA Detection
100.2. Add ~200 mL 1X wash buffer to each of two wash trays
SECTION: Protein Blocking
102. Incubate with Protein Block 20 min Room temperature.

 

SECTION: Protein Blocking
104. Submerge slide holder in fresh PBS-T 2 min Room temperature.
SECTION: Protein Blocking
105. Submerge slide holder in fresh PBS-T 2 min Room temperature.
SECTION: Protein Blocking
106.1. Discard RNA detection reagents. 
SECTION: Protein Blocking
106.2. Add ~200 mL PBS-T to each of two wash trays.

 

SECTION: Protein Detection
109.  Incubate with diluted secondary antibody 60 min Room temperature.

 

SECTION: Protein Detection
110. Remove slide holder from humidifying tray & decant (without removing slides from holder).
SECTION: Protein Detection
111. Submerge slide holder in fresh PBS-T 2 min Room temperature.
SECTION: Protein Detection
112. Submerge slide holder in fresh PBS-T 2 min Room temperature
SECTION: Protein Detection
113.1. Discard protein blocking reagents.
SECTION: Protein Detection
113.2. Add ~200 mL PBS-T to each of two wash trays
SECTION: Nuclei Staining and Cover Slipping
114.1. Apply DAPI 30 s Room temperature.
SECTION: Nuclei Staining and Cover Slipping
114.2. Decant slide to remove DAPI.
SECTION: Nuclei Staining and Cover Slipping
114.3. Mount slides by adding 2-4 drops of ProLong Gold antifade mounting media to each slide, followed by application of a #1.5 cover glass. 

 

SECTION: Nuclei Staining and Cover Slipping
115. Place slides flat in a dry, dark space to air dry 30 min Room temperature. 
SECTION: Nuclei Staining and Cover Slipping
116. Store at 4 °C and image within two weeks.