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Anti-PD-L1[clone 28-8]

Biogenex i6000 with offline heat

protocols for automated
immunohistochemistry

induced epitope retrieval (HEIR)

Anti-PD-L1[clone 28-8]
protocols for automated
immunohistochemistry IHC reagents

 Xylene (EM Science, cat# UN1307)

 Ethanol (AAPER alcohol and Chemical Co)
 Wash Buffer (DAKO, cat# S3006)
 Target Retrieval Solution, 10X (Dako, cat# S1699)
 Novolink Max Polymer Detection kit (Leica, cat# RE7260-CE)
 Peroxidase block (Dako cat# S2003)
 Protein block (Dako cat# X0909)
 Common antibody Diluent (BioGenex, cat# HK156-5K)
 DAB (DAKO cat# K3468)
 Cytoseal mounting medium (Richard-Allen Scientific)
 Anti PD-L1 antibody [28-8] (Abcam, cat# ab205921, final concentration 2
µg/mL)
 Isotype control rabbit monoclonal antibody (Abcam, cat# ab172730, final
concentration 2 µg/mL)

Equipment

 Leica ST5020 Multistainer

 BioGenex i6000 Autostainer
 BioCare Medical Decloaking Chamber™ Plus
 Leica Auto Coverslipper CV5030

HIER method

1. Perform deparaffinization and rehydration on Leica ST5020 Multistainer using

the following program:
a. 2 x 5 min wash with xylene
b. 2 x 2 min wash with 100% ethanol
c. 2 x 2 min wash with 95% ethanol
d. 2 x 2 min wash with 70% ethanol
e. 1 x 2 min was with dH2O
2. Perform antigen retrieval on Biocare Medical Decloaking Chamber™ Plus
using the following setting
a. Target Retrieval Solution, heated to 110°C (P1) for 10 min
b. move to the next step with P2 FAN ON at 95°C and FAN OFF at 90°C
3. Cool slide at room temperature (RT) for 15 min. Rinse with dH2O ~ 1 minute

IHC method

1. Set up slides on BioGenex i6000 Autostainer

2. Apply the peroxidase block for 10 min
3. Wash 3 x with wash buffer

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Anti-PD-L1[clone 28-8] 4. Apply the protein block to the slides and incubate 20 min at room
protocols for automated temperature (RT)
immunohistochemistry 5. Wash 3 x with wash buffer
6. Apply the pre-diluted antibodies to the slides (see Reagents) and incubate for
1 hour at RT
7. Wash 3 x with wash buffer
Anti-PD-L1[clone 28-8] 8. Add the post primary block (NovoLink Kit) to the slides and incubate for 30
protocols for automated min
immunohistochemistry 9. Wash 3X with IHC wash buffer
10. Add the NovoLink polymer (NovoLink Kit) to the slides, incubate for 45 min
11. Rinse 3 x with wash buffer
12. Add the DAB chromogen substrate and develop for 5 min
13. Wash slides 5 x with dH2O at RT
14. Counterstain with hematoxylin (Novolink kit) for 1 min at RT
15. Wash slides 5 x with dH2O RT

De-hydration and coverslipping

1. Remove the slides from BioGenex i6000 Autostainer

2. Set up slides on Leica Multistainer ST5020
3. Dehydrate using the following program:
a. 1 x 2 min wash with 70% ethanol
b. 1 x 2 min wash with 95% ethanol
c. 3 x 2 min wash with 100% ethanol
4. 3 x 2 min wash with xylene
5. Coverslip with Cytoseal Mounting Medium in the Leica Auto Coverslipper
CV5030

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 Leica BOND RX with on-line HIER
Anti-PD-L1[clone 28-8]
protocols for automated
immunohistochemistry

IHC reagents

Anti-PD-L1[clone 28-8] • Bond™ Dewax Solution (Leica, cat# AR9222)

protocols for automated • Bond™ Epitope Retrieval 1 (ER1) (Leica, cat# AR9961)
immunohistochemistry • Bond™ Wash Solution (Leica, cat# AR9590)
• Bond™ Polymer Refine Detection kit (Leica, cat# DS9800)
• Common Antibody Diluent (BioGenex, cat# HK156-5K)
• Cytoseal mounting medium (Richard-Allen Scientific)
• Anti PD-L1antibody [28-8] (Abcam, cat# ab205921) final concentration 2
µg/mL
• Isotype control rabbit monoclonal antibody (Abcam, cat# ab172730) final
concentration 2 µg/mL

Equipment

• Leica BOND RX autostainer

• Leica ST5020 Multistainer
• Leica coverslipper CV5030

Method

1. Heat and De-wax on Leica BOND RX autostainer

2. Performs antigen retrieval on Leica BOND RX autostainer at 100°C for 30
min using ER1 (Citra buffer, pH6 from Leica)
3. Apply the peroxidase block (Bond™ Polymer Refine Detection kit) for 10
min and then rinse 3 x with wash buffer
4. Apply the diluted antibodies to the slides (see Reagents) and incubate for
1 hour at room temperature (RT)
5. Wash 3 x with wash buffer
6. Add the post primary block (Bond™ Polymer Refine Detection kit) to the
slides, incubate for 30 min
7. Wash 3 x with wash buffer
8. Add the NovoLink polymer (Refine Kit) to the slides, incubate for 30 min
9. Wash 3 x with wash buffer
10. Add the DAB chromogen substrates (Refine Kit), Develop 10 min
11. Wash slides 5 x with dH2O at RT
12. Counterstain with hematoxylin (Refine Kit) for 8 min at RT
13. Wash slides 5 x with dH2O at RT

De-hydration and coverslipping

1. Remove the slides from Leica BOND RX autostainer

2. Set up slides on Leica ST5020 Multistainer
3. Dehydrate using following program:
a. 1 x 2 min wash with 70% ethanol
b. 1 x 2 min wash with 95% ethanol
c. 2 x 2 min wash with 100% ethanol
4. 2 x 2 min wash with xylene
5. Coverslip with Cytoseal Mounting Medium in Leica coverslipper CV5030

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 Ventana Ultra with on-line HIER
Anti-PD-L1[clone 28-8]
protocols for automated
immunohistochemistry
protocol
Anti-PD-L1[clone 28-8]
protocols for automated IHC reagents
immunohistochemistry • Xylene (EM Science, cat# UN1307)
• Ethanol (AAPER alcohol and Chemical Co)
• Universal HIER antigen retrieval reagent (Abcam, cat# ab208572)
• Antibody Diluent (Abcam, cat# ab64211)
• ChromoMap DAB kit (Ventana, cat# 760-159)
• Cytoseal mounting medium (Richard-Allen Scientific)
• Anti-Rabbit HQ (Ventana, cat# 760-4815)
• Anti-HQ HRP (Ventana, cat# 760-4820)
• Hematoxylin II (Ventana, cat# 790-2208)
• Bluing Reagent (Ventana, cat# 760-2037)
• Anti PD-L1 antibody [28-8] (Abcam, cat# ab205921) final concentration at
7.5 µg/mL

Equipment

• Ventana Ultra
• Leica ST5020 Multistainer
• BioCare Medical Decloaking Chamber™ Plus

IHC method

1. Bake and deparaffinize on Leica ST5020 Multistainer using the following

program:
a. Bake at 65°C for 10 min
b. 3 x 3 min with xylene
c. 2 x 2 min with 100% ethanol
d. 1 X 1 min with 95% ethanol
e. 1 X 1 min with 70% ethanol
f. 1 X 3 min with PBS
a. Perform antigen retrieval on BioCare Medical Decloaking Chamber™
Plus using the Universal HIER antigen retrieval reagent (Abcam,
ab208572) at 110°C for 10 min
2. Load slides on Ventana Ultra (setup protocol as listed below)

Protocol on Ventana Ultra

Use the following settings on Ventana Ultra:

1. Select – Antibody
2. Select – 1st Antibody Manual Application
3. Warm up slide to 37°C from Very Low Temperatures (Primary Antibody)
4. Hand apply (Primary Antibody) and incubate for 60 min
5. Select – Linking Antibody
6. Select – 2nd Antibody
7. Warm up slide to 37°C from Very Low Temperatures (2nd Antibody)
8. Apply one drop of Anti-Rabbit HQ (Detection #1) and incubate for 16 min
9. Select – Enzyme conjugate
10. Apply one drop of Anti-HQ HRP (Conjugate #1) and incubate for 16 min
11. Select – DAB
12. Select – Counterstain
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Anti-PD-L1[clone 28-8] 13. Select – Use RB for Counterstain
protocols for automated 14. Apply one drop of Hematoxylin II (Counterstain), and incubate for 8 min
immunohistochemistry 15. Select – Post Counterstain
16. Select – Use RB for Post Counterstain
17. Apply one drop of bluing reagent (Post Counterstain) and Incubate for 4 Min

Anti-PD-L1[clone 28-8] De-hydration and coverslipping

protocols for automated
immunohistochemistry 1. Remove the slides from Ventana Ultra
2. Wash slides with dawn soap
3. Rinse with deionized water
4. Set up slides on Leica ST5020 Multistainer and use the following program:
a) Dehydrate with 70% ethanol for 2 min
b) 95% ethanol for 2 min
c) 3 x 2 min with 100% ethanol
d) 3 x 2 min rinse with xylene
5. Coverslip with Cytoseal Mounting Medium manually

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 Dako Omnis
Anti-PD-L1[clone 28-8]
protocols for automated
immunohistochemistry

Primary antibody: Ab205921, clone 28-8

Anti-PD-L1[clone 28-8] Deparaffinization

protocols for automated
immunohistochemistry
Two-Phase- Solvent: Transport Liquid: Temperature Incubation Incubation # cycles: 1
Deparaffinization Clearify DI Water : 25° C (top): (bottom):
-IHC Clearing 10 seconds 1 minute
Agent
Two-Phase- Reagent: Incubation: # cycles: 1
Deparaffinization DI Water 5 seconds
-Wash-IHC

Antigen-retrieval (demasking)

Demasking-IHC Reagent: Temperature: 97°C Incubation: 30 cooling liquid:

EnVision min DI water
FLEX TRS,
Low pH

Staining

Step Reagent: Incubation: # cycles:

Wash Wash-Buffer 2:40 min 2
Primary Antibody PD-L1 (1:400) 1 hour
Wash Wash-Buffer 2 min 10
Endogenic Enzyme EnV FLEX Peroxidase-
Blocking Blocking Reagent 3 min
Wash Wash-Buffer 2 min 10
EnV FLEX + Rabbit
Secondary Reagent Linker 10 min
Wash Wash-Buffer 2 min 10
Labeled Polymer EnV FLEX/HRP 20 min
Wash Wash-Buffer 2 min 10
Wash Wash-Buffer 2 min 10
Wash DI Water 31 seconds 1
Wash Wash-Buffer 2 min 10
EnV FLEX Substrate
Substrate-Chromogen Working Solution 5 min
Wash Wash-Buffer 2 min 10
Wash DI Water 31 seconds 1
Wash Wash-Buffer 2 min 10
Counter-Staining

Staining Hematoxylin 3 min

Wash Wash-Buffer 2 min 10
Wash Wash-Buffer 2 min 10