Immunohistochemistry to Study the Relationship Between Macrophages and Infiltrated Immune Cells

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 formalin immediately for 24 h, divided into 3 to 4 mm sections, and embedded in paraffin, paying careful attention to the proper alignment of tissue in the block which is then stored at room temperature. Specimen selection is performed after verifying the medical records. The diagnosis is based on the American College of Rheumatology 1990 criteria which requires that subjects fulfill 3 of 5 domains: age >50 years, new headache, temporal artery tenderness, an erythrocyte sedimentation rate >50 mm/hour by Westergren method, and an abnormal TAB. For safety, protective gloves must be worn at all times when handling specimens, chemicals, and antibodies.

1. From the embedded blocks, cut 4-5 µm thick sections using a microtome. and stain with Hematoxylin and Eosin (H&E). Use a control section of selected normal tissue for quality assurance.
2. Float cut sections on a warm water bath heated to 40 °C to remove wrinkles and pick them up with a coated glass microscopic slide.  Place the glass slides on a warm plate in a 60 °C oven for 60 minutes to facilitate drying and adherence of the section to the slide.
3. Place the glass slides on a warm plate in a 60 °C oven for 60 minutes to facilitate drying and adherence of the section to the slide.
4. Mount 3 sections on microscope slides.
5. Place slides in a vertical rack and dry overnight at 37 °C for 24 h.
6. Place slides in a container and store at 4 °C.

2. Immunohistochemical Preparation, Dewaxing, Antigen Retrieval and Staining

1. Load slides into a slide rack. Run the rack through dishes as follows: Twice in 100% xylene for 5 minutes with 10 seconds of gentle agitation every 30 seconds, once in 100% ethanol with 10 seconds agitation, once in 90% ethanol with 10 seconds agitation, once in 70% ethanol with 10 seconds agitation, and twice in double distilled H₂O with 10 seconds agitation.        
   NOTE: Handling of xylene and acetone should be done in ventilated hoods to avoid inhalation and respiratory toxicity.
2. Retrieve the antigen by transferring the rack into a glass container with 200 mL of 10 mmol/L, pH 6.0 citrate buffer prewarmed to 95 °C. Set the timer for 30 minutes in the water bath, then cool at room temperature for 20 minutes then rinse with running water for 5 minutes.
3. Remove endogenous peroxidase activity by incubating in 200 mL of 3% hydrogen peroxide for 10 minutes. Wash slides 3 times in 200 µL of Tris-buffered saline solution (TBSS).
4. Remove the slides from the rack and dab each one gently to wipe out excess buffer. For staining, add 200 µL of the following primary antibodies, add coverslips on slides, and incubate in a humid chamber for 1 hour at room temperature:
   Anti-folate receptor beta (FRB) antibody diluted 1:800  
   Monoclonal mouse anti-human CD68, 1:200 dilution  
   Polyclonal rabbit anti-CD3 1:100 dilution
   NOTE: Formalin-fixed paraffin-embedded sections of the placenta were also processed as outlined in Steps 2.1 to 2.9, and incubated with anti-FRB18, and used as a positive control. Staining results were obtained by finding the optimal antibody concentration and was performed by titrating the antibody in double dilutions (e.g., 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600).
5. Remove the coverslip after incubation and discard. Rinse slides 3 times for 2 minutes each with TBSS, drain, and wipe excess buffer.
6. Add 200 µL of secondary antibody solution containing biotinylated Anti-Rabbit/Mouse secondary antibody to react with unconjugated primary antibody bound to tissue antigen. Place coverslips on slides and incubate in a humid chamber for 45 minutes at room temperature.
7. Remove the coverslip after incubation and discard. Rinse slides 3 times for 2 minutes each with TBS, drain, and wipe excess buffer.
8. Add 200 µL of diaminobenzidine (DAB)+substrate buffer (Imidazole HCl) -chromogen system and incubate for 10 minutes to visualize the staining pattern. Wash with TBSS then in running tap water for 10 minutes.
9. Counterstain with hematoxylin for 3 minutes.
10. Mount the slides by applying 70 µL of mounting medium to the surface of the slide. Slowly tip the coverslip onto the mounting medium and avoid creating bubbles as you lower it into place. Wait 24 hours to dry. 

3. Histopathologic and Immunohistochemical (IHC) Analysis

Examine the H&E and IHC stained sections from GCA-positive and normal subjects using a light microscope.

1. For the H&E stained sections
   1. Assess the vascular architecture and identify endothelial cells in the tunica intima, smooth muscle cells in the tunica media, and fibroblasts and vasa vasorum in the tunica adventitia.
   2. Identify GCA features, which include lymphocyte and macrophage/histiocyte infiltration, cellular hyperplasia, and internal elastic lamina degradation.
   3. Analyze the lymphohistiocytic infiltrate by identifying and quantifying the number of macrophages relative to the lymphocytes. Assess the extent of internal elastic lamina disruption and hyperplastic changes in resident cells and compare them with controls.
2. For the IHC stained sections
   1. Examine the staining pattern of FRB, taking note of its expression by a particular type or types of cells and its distribution within the vascular layers and its relationship to the total immune infiltrate, the total macrophage marker, anti-CD68, and the pan lymphocyte marker anti CD3.
   2. Quantify the expression of FRB, CD68, and CD3 cells by counting the positively stained cells on 10 multiple randomly selected high power fields (hpf) and record the means.        
      NOTE: Identification of these normal and pathologic cells and structures was performed by a certified cardiovascular pathologist and rheumatologist, and the results were recorded for all cases.
   3. Capture representative images using a digital camera.
      NOTE: After these steps, remaining aliquots may be stored at -80 °C.