Automated Immunohistochemical Staining Protocol for Anti-PD-L1
Licia Miller Product Manager
Phase 1 Deparaffinization and antigen retrieval
- Ventana Ultra
Required Materials
IHC reagents
- Xylene
- Ethanol
- HIER Antigen Retrieval Universal Reagent
Equipment
- Leica ST5020 Multistainer
- BioCare Medical Decloaking Chamber™ Plus
Experimental Steps
1. Perform sample baking and dewaxing on a Leica ST5020 Multistainer according to the following procedure :
• 3 × 3 min, xylene
• 2 × 2 min, 100% ethanol
• 1 × 1 min, 95% ethanol
• 1 × 1 min, 70% ethanol
• 1 × 3 min , PBS treatment
• Bake at 65°C for 10 minutes
2. Perform antigen retrieval using HIER Antigen Retrieval Universal Reagent at 110°C for 10 minutes in a BioCare Medical Decloaking Chamber™ Plus.
- Dako Omnis
Required Materials
- Primary antibody: anti-PD-L1 antibody
- Clearify Clearing Agent
- Deionized water
- EnVision FLEX TRS, Low pH
Experimental Steps
1. Dewaxing.
Phase 1:
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Phase 2:
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2. Antigen retrieval (demasking).
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Phase 2 IHC staining
- Leica BOND RX
Required Materials
IHC Reagents
- Bond™ Dewaxing Solution
- Bond™ Epitope retrieval 1 (ER1)
- Bond™ Cleaning Fluid
- Bond™ Polymer Refine Detection Kit
- Commonly used antibody diluents
- Anti-PD-L1 antibodies
- Isotype control antibody, such as rabbit monoclonal antibody
Equipment
- Leica BOND RX A utostainer
Experimental Steps
1. Warm and dewax in a Leica BOND RX automatic stainer.
2. Perform antigen retrieval in a Leica BOND RX Autostainer using ER1 (Leica's Citra buffer, pH 6) at 100°C for 30 minutes.
3. Incubate with peroxidase blocking reagent (Bond™ Polymer Refine Detection Kit) for 10 minutes, then rinse three times with wash buffer.
4. Apply the diluted antibody on the slide and incubate at room temperature for 1 hour.
5. Wash 3 times with wash buffer.
6. Add primary antibody post-blocking solution (Bond™ Polymer Refine Detection Kit) to the slides and incubate for 30 minutes.
7. Wash 3 times with wash buffer.
8. Add NovoLink polymer (Refine Kit) to the slide and incubate for 30 minutes.
9. Wash 3 times with wash buffer.
10. Add DAB chromogenic substrate (Refine Kit) and develop color for 10 minutes.
11. Wash the slides 5 times with dH2O at room temperature.
12. Counterstain with hematoxylin (Refine Kit) for 8 minutes at room temperature.
13. Wash the slides 5 times with dH2O at room temperature.
- Ventana Ultra
Required Materials
IHC reagents
- Antibody diluent
- ChromoMap DAB Kit
- Anti-rabbit HQ
- Anti-HQ HRP
- Hematoxylin II
- Bluing reagent
- Anti-PD-L1 antibodies
Equipment
-Ventana Ultra
Experimental Steps
1. Load the slides onto the Ventana Ultra.
2. Select - Antibody.
3. Selection - Manual application of primary antibody.
4. Warm the slides from cold to 37°C (primary antibody).
5. Manually add primary antibody and incubate for 60 minutes.
6. Select - Linking Antibody.
7. Select - the secondary antibody.
8. Warm the slides from cold to 37°C (secondary antibody).
9. Add a drop of high-quality anti-rabbit secondary antibody (Detection #1) and incubate for 16 minutes.
10. Selection - enzyme conjugate.
11. Add one drop of Anti-HQ HRP (Conjugate #1) and incubate for 16 minutes.
12. Select -DAB.
13.Select - Restain.
14. Optional - Counterstain with RB.
15. Add one drop of Hematoxylin II (counter stain) and incubate for 8 minutes.
16. Selection - post-counter-staining.
17. Optional - Use RB for Post Counterstain.
18. Apply a drop of blueing reagent (Post Counterstain) and incubate for 4 minutes.
- Dako Omnis
Required Materials
-Primary antibody: PD-L1 antibody
- Washing buffer
- EnV FLEX Peroxidase Blocking Reagent
- EnV FLEX + Rabbit Linker
- Labeled polymer EnV FLEX/HRP
- EnV FLEX Substrate Working Solution
-Hematoxylin
-Deionized water
Experimental Steps
1. Wash twice with wash buffer, 2 minutes and 40 seconds each time.
2. Incubate with primary antibody (PD-L1, 1:400) for 1 hour.
3. Wash 10 times with wash buffer, 2 minutes each time.
4. Endogenous enzyme blocking: Block with EnV FLEX peroxidase blocking reagent for 3 minutes.
5. Wash 10 times with wash buffer, 2 minutes each time.
6. Incubate the secondary antibody (EnV FLEX + Rabbit Linker) for 10 minutes.
7.10 × 2 min with wash buffer.
8. Incubate the labeled polymer (FLEX/HRP) for 20 minutes.
9. Wash.
• 10 × 2 min, wash buffer
• 10 × 2 min, wash buffer
• 1 × 31 sec, deionized water
• 10 × 2 min, wash buffer
10. Incubate the substrate-chromogen (EnV FLEX Substrate Working Solution) for 5 minutes.
11. Wash.
• 1 × 31 sec, deionized water
• 10 × 2 min, wash buffer
12. Incubate with counterstain (hematoxylin) for 3 minutes.
13. Wash.
• 10 × 2 min, wash buffer
• 10 × 2 min, wash buffer
Phase 3 Dehydration and coverslipping
- Leica BOND RX
Required Materials
IHC reagents
- Ethanol
- Xylene
- Cytoseal Mounting Medium
Equipment
- Leica ST5020 Multistainer
- BioGenex i6000 Autostainer
- Leica Auto Coverslipper CV5030
Experimental Steps
1. Remove the slides from the Leica BOND RX autostainer.
2. Mount the slides onto the Leica ST5020 Multistainer.
3. Dehydrate with ethanol.
• 1 × 2 min wash with 70% ethanol
• 1 × 2 min wash with 95% ethanol
• 1 × 2 min wash with 95% ethanol
4. Wash twice with xylene, 2 minutes each time.
5. Place the coverslip with Cytoseal mounting medium into the Leica Auto Coverslipper CV5030.
- Ventana Ultra
Required Materials
IHC reagents
-Xylene
-Ethanol
-Deionized water
- Cytoseal Mounting Medium
Equipment
-Ventana Ultra
-Leica ST5020 Multistainer
Experimental Steps
1. Remove the slides from the Ventana Ultra.
2. Wash the slides with soap first , then rinse with deionized water.
3. Mount the slides in a Leica ST5020 Multistainer and use the following procedure.
• Dehydrate with 70% ethanol for 2 minutes
• 95% ethanol for 2 minutes
• 100% ethanol treatment, 3 × 2 minutes
• Rinse in xylene 3 times, 2 minutes each
4. Manually mount the slides with Cytoseal mounting medium and coverslips.
For more product details, please visit Aladdin Scientific website.