An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis

This immunohistochemical and histopathologic protocol can be used to examine folate receptor beta, which is a candidate protein with potential prognostic and therapeutic value to the vascular microenvironment in giant cell arteritis. This protocol is time-tested and widely employed and can be used to explore the folate receptor beta attributes in temporal artery tissues, even after long-term storage. To acquire tissue sections, use a microtome to slice four to five micrometer thick sections of paraffin-embedded temporal arteries, floating each section in a 40 degree Celsius water bath as they are obtained, to remove wrinkles.

Use glass microscope slides to capture three sections per slide, and warm the slides on a warming plate, in a 60 degree Celsius oven for 60 minutes. When the sections have adhered to the slides, place the slides into a vertical rack for drying at 37 degrees celsius for 24 hours. The next day, transfer the slides to a slide container for storage at four degrees Celsius for at least 12 hours.

Next, load slides into a slide rack and hydrate the tissues with two five-minute, 100%xylene immersions and a descending, five-minute per concentration ethanol immersion series, with 10 seconds of agitation every 30 seconds. After the 70%ethanol immersion, rinse the slides two times in double-distilled water for five minutes, with 10 seconds of agitation every 30 seconds. For antigen retrieval, transfer the rack into a glass container with 200 microliters of 95 degrees Celsius, 10 millimolar per liter citrate buffer for 30 minutes.

At the end of the incubation, allow the slides to cool to room temperature for 20 minutes before rinsing with running water for five minutes. To remove the endogenous peroxidase activity, first use a hydrophobic marker to draw a circle around each tissue sample, before incubating the samples in 200 microliters of 3%hydrogen peroxide for 10 minutes, followed by 3 washes in 200 micoliters of tris-buffered saline solution, or TBSS, per wash. After the last wash, dab each slide gently to remove any excess buffer and add 200 microliters of the primary antibody cocktail of interest and a cover slip to each slide for a one-hour incubation in a humid chamber at room temperature.

At the end of the incubation, discard the cover slips and rinse the slides with three two-minute washes in fresh TBSS. After removing the excess buffer, add 200 microliters of an appropriate biotinylated secondary antibody solution and a cover slip to each slide for a 45-minute incubation in the humid chamber at room temperature. After discarding the cover slips, rinse the slides with TBSS as demonstrated.

Remove any excess buffer. And label the sections with 200 microliters of DAB substrate buffer for 10 minutes at room temperature. At the end of the incubation, wash the slides with TBSS and running tap water for 10 minutes, before counterstaining with hematoxylin for three minutes.

Then, add 70 microliters of an appropriate mounting medium to each slide and slowly tip a glass cover strip onto the mounting medium to avoid creating bubbles as the cover slip is lowered into place. For histopathologic analysis, place the slide onto the stage of a light microscope and assess the vascular architecture of the first tissue section. Then, quantify the number of macrophages relative to the number of lymphocytes per section.

For immunohistochemical analysis, examine the folate receptor beta staining pattern on each tissue section and quantify the number of folate receptor beta positive, CD68 positive, and CD3 positive cells in 10 randomly selected high power fields per section. Hematoxylin and eosin staining in normal specimens reveals a normal arterial anatomy, with endothelial cells in the tunica intima, smooth muscle cells in the tunica media, and a heterogeneous collagen matrix, that include fibroblasts and feeder vessels called vasa vasorum in the tunica adventitia. Giant cell arteritis positive temporal arteries demonstrate a moderate to severe inflammation, with aggregates and macrophages, or histiocytes, lymphocytes, occasional plasma cells, and multi-nucleated giant cells.

Luminal narrowing is also noted, and accompanied by intimal thickening and a 90%disruption of the internal elastic lamina. CD3 positive lymphocytes and CD68 positive macrophages are also present in all giant cell arteritis positive specimens, with similar staining patterns observed in all of the biopsies. Staining for folate receptor beta is restricted to macrophages and multi-nucleated giant cells that preferentially localize to the adventitia and media.

Notably, folate receptor beta comprised approximately 30%of the total macrophages. The temporal artery biopsy processing, cutting, and dewaxing require careful dexteriety in order to perform subsequent steps. The slide interpretation requires the expertise of an experienced cardiovascular pathologist.

Remind the viewers to use contact and respiratory precautions, such as gloves and ventilated hoods, when handling specimens and the reagents.