Immunohistochemistry to Study the Relationship Between Macrophages and Infiltrated Immune Cells

Begin with a slide containing formalin-fixed, deparaffinized, and rehydrated temporal artery biopsy sections with infiltrated immune cells, including lymphocytes and macrophages.

Activated macrophages express folate receptor beta or FR-β — a disease-related marker, differentiating the macrophages from other immune cells.

Transfer the slide into a pre-warmed buffer to retrieve surface receptors.

Treat the section with hydrogen peroxide, blocking intracellular peroxidase activity.

Introduce a mix of primary antibodies targeting CD3, CD68, and FR-β, which bind to immune cells expressing these proteins.

Add biotinylated secondary antibodies, interacting with primary antibodies, and wash.

Overlay with streptavidin-conjugated enzymes that bind with biotin.

Treat with a chromogenic substrate that interacts with enzymes, producing localized brown precipitates.

Introduce a counterstain — hematoxylin, staining the nuclei blue.

Microscopically identify small, round, brown-stained cells as CD3-expressing lymphocytes and large, irregular, brown-stained cells as CD68 and FR-β-expressing macrophages.

Quantify macrophages in the section to find their ratio amongst the total infiltrated immune cells.