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Automated IHC protocols for anti-PD-L1

Step-by-step protocols for automated IHC with Anti-PD-L1 [clone 28-8] using Biogenex i6000, LEICA BOND RX, Ventana Ultra, and Dako Omnis.
Last edited Tue 16 May 2023

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

1

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
2

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.
3

Cool slide at room temperature for 15 min.

  • Rinse with dH2O ~ 1 minute.

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] (Abcam, cat# ab205921, final concentration 2 µg/mL)
  • Isotype control rabbit monoclonal antibody (Abcam, cat# ab172730, final concentration 2 µg/mL)

Equipment

  • BioGenex i6000 Autostainer

Steps

1

Set up slides on BioGenex i6000 Autostainer.

2

Apply the peroxidase block for 10 min.

3

Wash 3 times with wash buffer.

4

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

5

Wash 3 times with wash buffer.

6

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

7

Wash 3 xtimes with wash buffer.

8

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

9

Wash 3 times with IHC wash buffer.

10

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

11

Rinse 3 times with wash buffer.

12

Add the DAB chromogen substrate and develop for 5 min.

13

Wash slides 5 times with dH2O at room temperature.

14

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

15

Wash slides 5 times with dH2O at room temperature.

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

1

Remove the slides from BioGenex i6000 Autostainer.

2

Set up slides on Leica Multistainer ST5020.

3

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
4

Wash with xylene, 3 times for 2 min.

5

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