Automated immunohistochemistry (IHC) protocols streamline staining workflows, ensuring reproducibility and efficiency in high-throughput labs. This guide focuses on PD-L1 detection using automated platforms such as Biogenex i6000, Leica BOND RX, Ventana Ultra, and Dako Omnis. Some platforms, such as the Ventana BenchMark ULTRA, offer full automation for high-throughput and standardized workflows. The protocol covers critical steps, including deparaffinization, antigen retrieval, antibody incubation, and chromogen development. Automation minimizes human error, standardizes results, and accelerates turnaround times compared to manual staining. By integrating optimized reagents and validated workflows, researchers can achieve consistent, high-quality staining for diagnostic and research applications. This resource is ideal for laboratories seeking to improve throughput while maintaining accuracy in biomarker detection.

Introduction

Immunohistochemistry is a cornerstone technique for visualizing protein expression in tissue samples. Automated IHC systems have revolutionized this process by reducing variability and increasing efficiency. Unlike manual staining, automation ensures precise reagent application, controlled incubation times, and consistent results across batches. These automated systems are designed to streamline laboratory workflows, integrating multiple functions to ensure reliable and standardized results across various platforms. This protocol demonstrates automated PD-L1 staining using multiple platforms, providing detailed steps for antigen retrieval, blocking, and chromogen development. By leveraging automation, laboratories can meet growing demands for biomarker analysis in oncology, immunology, and translational research. The following sections outline principles, applications, and troubleshooting strategies to optimize automated IHC workflows.

Background and principles

Automated IHC builds on the principle of antibody-antigen binding, visualized through chromogenic reactions. Formalin-fixed paraffin-embedded (FFPE) tissues often require antigen retrieval to restore epitope accessibility, typically achieved via heat-induced epitope retrieval (HIER). Automated platforms replicate manual steps, such as deparaffinization, blocking, and antibody incubation, while ensuring precision and reproducibility. Key advantages include reduced operator variability and improved standardization, critical for clinical and research settings. By integrating validated reagents and optimized protocols, automated IHC enhances sensitivity and specificity, enabling accurate detection of biomarkers such as PD-L1. The choice of detection system and careful optimization of staining intensity are crucial for accurate biomarker detection while preserving tissue morphology. Understanding these principles ensures successful implementation and reliable staining outcomes.

Stage 1 - Deparaffinization and antigen retrieval

Materials required

IHC reagents

• Xylene (EM Science, cat# UN1307)
• Ethanol (AAPER alcohol and Chemical Co)
• Target Retrieval Solution, 10X (Dako, cat# S1699)

Equipment

• Leica ST5020 Multistainer
• BioCare Medical Decloaking Chamber™ Plus

Steps

Perform deparaffinization and rehydration on Leica ST5020 Multistainer using the following program.

• 2 x 5 min wash with xylene
• 2 x 2 min wash with 100% ethanol
• 2 x 2 min wash with 95% ethanol
• 2 x 2 min wash with 70% ethanol
• 1 x 2 min was with dH₂O

Perform antigen retrieval on Biocare Medical Decloaking Chamber™ Plus using the following setting.

• Target Retrieval Solution, heated to 110°C (P1) for 10 min.
• Move to the next step with P2 FAN ON at 95°C and FAN OFF at 90°C.

Cool slide at room temperature for 15 min.

• Rinse with dH₂O ~ 1 minute.

Materials required

IHC reagents

• Xylene (EM Science, cat# UN1307)
• Ethanol (AAPER alcohol and Chemical Co)
• Universal HIER antigen retrieval reagent (eg ab208572)

Equipment

• Leica ST5020 Multistainer
• BioCare Medical Decloaking Chamber™ Plus

Steps

Bake and deparaffinize on Leica ST5020 Multistainer using the following program:

• Bake at 65°C for 10 min
• 3 x 3 min with xylene
• 2 x 2 min with 100% ethanol
• 1 X 1 min with 95% ethanol
• 1 X 1 min with 70% ethanol
• 1 X 3 min with PBS

Perform antigen retrieval on BioCare Medical Decloaking Chamber™ Plus using the Universal HIER antigen retrieval reagent (Abcam, ab208572) at 110°C for 10 min.

Materials required

• Primary antibody: ab205921, clone 28-8
• Clearify Clearing Agent
• DI water
• EnVision FLEX TRS, low pH

Steps

Two-Phase Deparaffinization-IHC

Solvent: Clearify Clearing Agent

Transport Liquid: DI Water

Temperature: 25° C

Incubation (top): 10 seconds

Incubation (bottom): 1 minute

# cycles: 1

Two-Phase Deparaffinization-Wash-IHC

Reagent: DI Water

Incubation: 5 seconds

# cycles: 1

Antigen-retrieval (demasking)

Reagent: EnVision FLEX TRS, Low pH

Temperature: 97°C

Incubation: 30 min

Cooling liquid: DI water

Stage 2 - IHC staining

Materials required

IHC reagents

• Wash Buffer (DAKO, cat# S3006)
• 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)
• Anti PD-L1 antibody [28-8] (ab205921, final concentration 2 µg/mL)
• Isotype control rabbit monoclonal antibody (ab172730, final concentration 2 µg/mL)

Equipment

• BioGenex i6000 Autostainer

Steps

Set up slides on BioGenex i6000 Autostainer.

Apply the peroxidase block for 10 min.

Wash 3 times with wash buffer.

Apply the protein block to the slides and incubate 20 min at room temperature.

Wash 3 times with wash buffer.

Apply the pre-diluted antibodies to the slides (see Materials) and incubate for 1 hour at room temperature.

Wash 3 times with wash buffer.

Add the post primary block (NovoLink Kit) to the slides and incubate for 30 min.

Wash 3 times with IHC wash buffer.

Add the NovoLink polymer (NovoLink Kit) to the slides and incubate for 45 min.

Rinse 3 times with wash buffer.

Add the DAB chromogen substrate and develop for 5 min.

Wash slides 5 times with dH₂O at room temperature.

Counterstain with hematoxylin (Novolink kit) for 1 min at room temperature.

Wash slides 5 times with dH₂O at room temperature.

Materials required

IHC reagents

• Bond™ Dewax Solution (Leica, cat# AR9222)
• Bond™ Epitope Retrieval 1 (ER1) (Leica, cat# AR9961)
• Bond™ Wash Solution (Leica, cat# AR9590)
• Bond™ Polymer Refine Detection kit (Leica, cat# DS9800)
• Common Antibody Diluent (BioGenex, cat# HK156-5K)
• 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

Heat and De-wax on Leica BOND RX autostainer.

Perform antigen retrieval on Leica BOND RX autostainer at 100°C for 30 min using ER1 (Citra buffer, pH6 from Leica).

Apply the peroxidase block (Bond™ Polymer Refine Detection kit) for 10 min and then rinse 3 times with wash buffer.

Apply the diluted antibodies to the slides (see Materials) and incubate for 1 hour at room temperature.

Wash 3 times with wash buffer.

Add the post primary block (Bond™ Polymer Refine Detection kit) to the slides and incubate for 30 min.

Wash 3 times with wash buffer.

Add the NovoLink polymer (Refine Kit) to the slides and incubate for 30 min.

Wash 3 times with wash buffer.

Add the DAB chromogen substrates (Refine Kit) and develop 10 min.

Wash slides 5 times with dH₂O at room temperature.

Counterstain with hematoxylin (Refine Kit) for 8 min at room temperature.

Wash slides 5 times with dH₂O at room temperature.

Materials required

IHC reagents

• Antibody Diluent (ab64211)
• ChromoMap DAB kit (Ventana, cat# 760-159)
• 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] (ab205921) final concentration at 7.5 µg/mL

Equipment

• Ventana Ultra

Steps

Load slides on Ventana Ultra.

Select - Antibody

Select - 1st Antibody Manual Application

Warm up slide to 37°C from very low temperatures (primary antibody)

Hand apply (Primary Antibody) and incubate for 60 min.

Select – Linking Antibody.

Select – 2nd Antibody.

Warm up slide to 37°C from very low temperatures (2nd antibody).

Apply one drop of Anti-Rabbit HQ (Detection #1) and incubate for 16 min.

Select - Enzyme conjugate

Apply one drop of Anti-HQ HRP (Conjugate #1) and incubate for 16 min.

Select - DAB

Select - Counterstain

Select - Use RB for Counterstain

Apply one drop of Hematoxylin II (Counterstain) and incubate for 8 min.

Select - Post Counterstain

Select - Use RB for Post Counterstain

Apply one drop of bluing reagent (Post Counterstain) and incubate for 4 min.

Materials required

• Primary antibody: PD-L1, ab205921, clone 28-8
• Wash buffer
• EnV FLEX Peroxidase Blocking Reagent
• EnV FLEX + Rabbit Linker
• Labeled Polymer EnV FLEX/HRP
• EnV FLEX Substrate Working Solution
• Hematoxylin
• DI water

Steps

Wash

• 2 x 2 min 40 sec with wash buffer

Incubate the primary antibody (PD-L1, 1:400) for 1 hour

Wash

• 10 x 2 min with wash buffer

Endogenic enzyme blocking.

Block for 3 min with EnV FLEX Peroxidase Blocking Reagent.

Wash.

• 10 x 2 min with wash buffer

Incubate the secondary reagent (EnV FLEX + Rabbit Linker) for 10 min.

Wash.

• 10 x 2 min with wash buffer

Incubate the labeled polymer (FLEX/HRP) for 20 min.

Wash.

• 10 x 2 min with wash buffer
• 1 x 31 sec with DI water
• 10 x 2 min with wash buffer

Incubate the substrate-chromogen (EnV FLEX Substrate Working Solution) for 5 min.

Wash.

• 10 x 2 min with wash buffer
• 1 x 31 sec with DI water
• 10 x 2 min with wash buffer

Incubate with the counterstain (hematoxylin) for 3 min.

Wash.

• 10 x 2 min with wash buffer

Stage 3 - De-hydration and coverslipping

Materials required

IHC reagents

• Xylene (EM Science, cat# UN1307)
• Ethanol (AAPER alcohol and Chemical Co)
• Cytoseal mounting medium (Richard-Allen Scientific)

Equipment

• Leica ST5020 Multistainer
• BioGenex i6000 Autostainer
• Leica Auto Coverslipper CV5030

Steps

Remove the slides from BioGenex i6000 Autostainer.

Set up slides on Leica Multistainer ST5020.

Dehydrate using ethanol.

• 1 x 2 min wash with 70% ethanol
• 1 x 2 min wash with 95% ethanol
• 3 x 2 min wash with 100% ethanol

Wash with xylene, 3 times for 2 min.

Coverslip with Cytoseal Mounting Medium in the Leica Auto Coverslipper CV5030.

Materials required

IHC reagents

• Ethanol
• Xylene
• Cytoseal mounting medium (Richard-Allen Scientific)

Equipment

• Leica BOND RX autostainer
• Leica ST5020 Multistainer
• Leica coverslipper CV5030

Steps

Remove the slides from Leica BOND RX autostainer.

Set up slides on Leica ST5020 Multistainer.

Dehydrate using ethanol.

• 1 x 2 min wash with 70% ethanol
• 1 x 2 min wash with 95% ethanol
• 2 x 2 min wash with 100% ethanol

Wash with xylene, 2 times for 2 min.

Coverslip with Cytoseal Mounting Medium in Leica coverslipper CV5030.

Materials required

IHC reagents

• Xylene (EM Science, cat# UN1307)
• Ethanol (AAPER alcohol and Chemical Co)
• Deionized water
• Cytoseal mounting medium (Richard-Allen Scientific)

Equipment

• Ventana Ultra
• Leica ST5020 Multistainer

Steps

Remove the slides from Ventana Ultra.

Wash slides with dawn soap.

Rinse with deionized water.

Set up slides on Leica ST5020 Multistainer and use the following program.

• Dehydrate with 70% ethanol for 2 min
• 95% ethanol for 2 min
• 3 x 2 min with 100% ethanol
• 3 x 2 min rinse with xylene

Coverslip with Cytoseal Mounting Medium manually.

Comparison to other methods

Manual IHC staining is labor-intensive, time-consuming, and prone to variability, requiring skilled technicians for consistent results. Automated IHC systems overcome these limitations by standardizing workflows, making processes more efficient, and reducing processing time and human error. While manual methods offer flexibility for small-scale experiments, automation excels in high-throughput environments, processing up to 60 slides in a few hours. Automated platforms also integrate quality control features, minimizing reagent waste and optimizing turnaround times. Automation supports routine practice in clinical and experimental pathology by ensuring standardized and reproducible results, which are essential for reliable tissue biomarker analysis. Although initial investment in automation can be significant, long-term benefits include improved reproducibility, reduced labor costs, and enhanced diagnostic accuracy. For labs handling large volumes, automation is the preferred approach.

Applications

Automated IHC is widely used in clinical diagnostics, translational research, and drug development. It enables precise detection of biomarkers such as PD-L1, HER2, and Ki-67, supporting cancer immunotherapy studies and prognostic evaluations. High-throughput automation is essential for pathology labs processing large sample volumes, ensuring rapid and standardized results. Automated IHC platforms enable the detection of multiple markers through multiplexed staining, supporting research and diagnostics in diseases such as breast cancer and lung cancer. These automated systems can integrate IHC and ISHtechniques for comprehensive analysis, allowing simultaneous assessment of protein and gene alterations. Research applications include tissue-based studies in oncology, neuroscience, and immunology, where accurate protein localization is critical.

Limitations

Despite its advantages, automated IHC has limitations. Initial setup costs for instruments and reagents can be high, making it less accessible for small labs. Platform-specific protocols may restrict flexibility, requiring validation for new antibodies or tissue types. When using different protocols, it is essential to validate them with appropriate negative controls and positive staining to ensure assay specificity. The inclusion of negative staining and negative controls is crucial for validating results and confirming the absence of non-specific binding or crosstalk. The combination of automated steps and proper controls, such as negative control and positive staining, supports reliable assay performance. Technical issues such as reagent compatibility and machine calibration can impact staining quality. Additionally, automation does not eliminate the need for skilled personnel to oversee workflows and troubleshoot errors. Certain complex or non-standard assays may still require manual intervention. Understanding these constraints helps laboratories plan effectively and maintain high-quality results in automated IHC workflows.

Troubleshooting

Common issues in automated IHC include weak staining, high background, and uneven signal. Weak staining often results from insufficient antigen retrieval, incorrect antibody concentration, or suboptimal staining intensity. Optimizing staining intensity as part of the staining procedure, by adjusting incubation parameters and validating antibody dilutions, can resolve this issue. A high background may indicate inadequate blocking or reagent contamination; ensure the proper use of protein and peroxidase blocks. Uneven staining can stem from poor slide preparation or machine calibration; regular maintenance and quality checks are essential. Always verify the integrity of reagents and follow platform-specific guidelines. Implementing these troubleshooting strategies within a standardized staining procedure ensures reliable, reproducible results across automated IHC experiments.