Immunohistochemistry to Study the Relationship Between Macrophages and Infiltrated Immune Cells

Abstract

Source: Albano-Aluquin, S. et al., An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis. J. Vis. Exp. (2019)

This video demonstrates a technique to quantify the relative ratio of macrophages among the total infiltrated immune cell populations in temporal artery biopsy. The immunohistochemical staining of a tissue section affected by autoimmune disease selectively stains the immune cells with specific receptors to visually differentiate them from the rest of the cells.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Histopathological Preparation, and Processing After Obtaining Temporal Artery Biopsy NOTE: The temporal artery biopsy (TAB) is performed under local anesthesia and sterile conditions by a certified surgeon. After surgically obtaining a 3 cm arterial section on the more affected side, the specimen is fixed in fo…

Materials

Microtome	Reichert-Jung
Plain and frosted microscope slides and cover slips	Fisher Scientific
Vertical rack	Fisher Scientific
Light microscope	Olympus BX60 microscope
Xylene
Acetone in distilled water			concentrations 100%, 90%, 70%
Tris-buffered saline solution (TBS)	Dako Wash BufferS3006
3% hydrogen peroxide in TBS
Diaminobenzidine (DAB)	Sigma Fast Tablet set
Chemical Permount  Mounting Medium	Fisher Scientific	SP-15-100
Harleco Gill's III Hematoxylin	Fiisher Scientific 23-750-019
Harleco Eosin Y 1% Alcoholic Stock Solution	Fisher Scientific	23-749-977
10 mM citric acid monohydrate (pH 6.0)
Polyclonal rabbit anti-folate receptor beta	Manohar Ratnam		optimized at 1:800
Monoclonal mouse anti-human CD68	Dako	M071801	optimized at 1:200
Polyclonal rabbit anti-CD3	Agilent Dako	A0452	optimized at 1:100
Polymer goat anti- rabbit/mouse secondary antibody	Dako
Placental tissue			for FRB positive control