May 21, 2025

Public workspaceCD8 Immunohistochemistry Staining

DOI
dx.doi.org/10.17504/protocols.io.kxygxqx54v8j/v1
  • Michael Fotheringham1,
  • Logan L Engle2
  • 1Queen's University;
  • 2Johns Hopkins University
Michael Fotheringham
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DOI: dx.doi.org/10.17504/protocols.io.kxygxqx54v8j/v1
Protocol Citation: Michael Fotheringham, Logan L Engle 2025. CD8 Immunohistochemistry Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxqx54v8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 09, 2025
Last Modified: May 21, 2025
Protocol Integer ID: 218008
Abstract
Slide staining is performed on the Leica BOND RX. For detailed instructions, refer to the Leica BOND RX user manual. General steps and user-configurable parameters are outlined here for reproducibility.
Materials

- 4-μm tissue sections

- Leica BOND RX (Leica Biosystems, Buffalo Grove, IL, USA; RRID:SCR_025548)

- Bond Polymer Refine Kit (DS9800, Leica Biosystems; RRID:AB_2891238)

- Anti-CD8 clone 4B11 (CD8-4B11-L-CE, Leica Biosystems; RRID:AB_10555292)

- EcoMount (5082832, Biocare Medical, Walnut Creek, CA, USA)

CD8 Immunohistochemistry Staining
CD8 Immunohistochemistry Staining
2h 45m
2h 45m
Obtain 4-μm sections of each tissue block.

Slide staining is performed on the Leica BOND RX (Leica Biosystems, Buffalo Grove, IL, USA; RRID:SCR_025548) using the Bond Polymer Refine Kit (DS9800, Leica Biosystems; RRID:AB_2891238).


Consult the user manual for your Leica machine for detailed instructions. User-configured parameters are detailed below:

2h 30m
Select Leica's on-line Bake and Dewax protocol.
Select high pH EDTA buffer antigen retrieval for 20 minutes at 100°C.
Select endogenous peroxidase blocking using the peroxidase block included in the Bond Polymer Refine Kit (Leica Biosystems).

Select anti-CD8 clone 4B11 (CD8-4B11-L-CE, Leica Biosystems; RRID:AB_10555292) for 15 minutes at a concentration of 19.2 μg/mL at room temperature.

Select counterstain with hematoxylin included in the Bond Polymer Refine Kit (Leica Biosystems).
Initiate the staining protocol on the Leica BOND RX (Leica Biosystems).
Remove slides from the Leica Bond RX and dehydrate through graded-alcohol. Coverslip using EcoMount (5082832, Biocare Medical, Walnut Creek, CA, USA).
15m
Protocol references
Taube JM, Akturk G, Angelo M, Engle EL, Gnjatic S, Greenbaum S, Greenwald NF, Hedvat CV, Hollmann TJ, Juco J, Parra ER, Rebelatto MC, Rimm DL, Rodriguez-Canales J, Schalper KA, Stack EC, Ferreira CS, Korski K, Lako A, Rodig SJ, Schenck E, Steele KE, Surace MJ, Tetzlaff MT, von Loga K, Wistuba II, Bifulco CB; Society for Immunotherapy of Cancer (SITC) Pathology Task Force. The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation. J Immunother Cancer. 2020 May;8(1):e000155. doi: 10.1136/jitc-2019-000155. Erratum in: J Immunother Cancer. 2020 Jun;8(1):e000155corr1. doi: 10.1136/jitc-2019-000155corr1. PMID: 32414858; PMCID: PMC7239569.
Acknowledgements
Logan L Engle, Michael Fotheringham