Reliable CD4 and CD8 T Cell Marker Immunohistochemistry on Formalin‐Fixed and Histochoice‐Fixed Paraffin Embedded Mouse Spleen
Authors: Bradshaw Kaitlynn Nicole , Weng‐Race Julin , Ward Jerrold M , Rehg Jerold E , Kovacs Joseph A , Davis Anne Sally 
Published: November 2023

CD4 and CD8 are glycoproteins found on the surface of T lymphocytes and other immune cells. Researchers have long been able to detect these T cell subset markers in formalin‐fixed, paraffin‐embedded human tissues. CD4+ and CD8+ T lymphocyte levels and distribution are commonly used to determine disease state, for example in HIV infections. To date, reliable and consistent identification of these markers by immunohistochemistry (IHC) in fixed, paraffin‐embedded mouse tissues has been difficult. Typically, researchers must rely on frozen mouse tissues in order to detect these T cell subset markers in mice. However, not all facilities can support frozen IHC work. In contrast, formalin‐fixed, paraffin‐embedded (FFPE) is the standard archival tissue format. Therefore, we focused on developing reliable CD4 and CD8 IHC for mouse tissues fixed in formalin or Histochoice (Sigma‐Aldrich; St. Louis, MO), an alcohol‐based fixative.

Normal mouse spleen was selected as the target tissue because it is a tissue in which a standard pattern for CD4 + and CD8 + T cell distribution would be recognizable. We used Anti‐Mouse CD8a Purified [4SM15] and Anti‐Mouse CD4 [4SM95] (eBiosciences; San Diego, CA) for which some prior success in IHC on FFPE had been reported by co‐author Rehg. For the FFPE tissues, we tried different heat antigen retrieval (AR) methods including citrate, TRIS and EDTA based buffers at multiple pHs. Because alcohol‐based fixatives do not induce the aldehyde cross‐linking and consequent epitope masking encountered with formalin fixation, AR was omitted for Histochoice‐fixed, paraffin‐embedded (HCPE) tissues. Multiple detection approaches were tried, including avidin‐biotin complex with horseradish peroxidase (ABC HRP) and polymer based detection.

In FFPE tissues, high (8.0) pH EDTA heat antigen retrieval provided the best results, which is not unexpected given that CD4 and CD8 are membranous markers. For CD4, the optimal primary []s were 1.6mg/ml for FFPE spleen and 12.5 mg/ml for HCPE. For CD8, the optimal primary concentration was 5 mg/ml in both the FFPE and HCPE spleen. Use of a secondary antibody, Biotin‐SP‐conjugated AffiniPure Donkey Anti‐rat IgG (Jackson ImmunoResearch; West Grove, PA) with an ABC HRP detection approach worked best for both FFPE and HCPE CD8 labeling as well as for the FFPE CD4 labeling. In contrast, the best detection system for the HCPE CD4 IHC was a polymer based detection approach, specifically ImmPRESS Anti‐rat IgG (Peroxidase) Polymer Detection Kit (Vector Labs; Burlingame, CA). These combinations provided the least background while still enabling easy visualization of the markers in the appropriate biological pattern. This work is being expanded to other mouse tissues including the lung where it will be used to examine changes in CD4 and CD8 T cell subsets in infectious disease studies.

Support or Funding Information

This research has been funded by Dr. Davis' startup funds from the Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University and the Intramural Research Program of the NIH Clinical Center (ZIA CL000037‐27, ZIA CL000146‐21, and ZIA CL000192‐17).