Analysis of nitrotyrosine on thymic sections.

(A) Nitrotyrosine and keratin double staining. Nitrotyrosine spots (red staining) are mainly observed in the cortex and at the corticomedullary junction (i). Double staining with antikeratin antibody delineates the cortex and the medulla (ii, iii). The superimposed microphotography is shown in panel Aiii (× 25). A higher magnification of the white rectangle (iv) shows that some epithelial cells (green staining) are often contained in the nitrotyrosine spots (× 100). The control performed by absorbing the antinitrotyrosine activity with competing nitrotyrosine is negative (v). The control performed by omitting the first layers is also negative (vi). (B) Nitrotyrosine and TUNEL double staining. Nitrotyrosine spots (red staining) in a cortical area (ii) are often associated with cells positive in a TUNEL reaction (green staining) (i, iii) (× 25). Double-stained cells are shown at a higher magnification of the white rectangle (iii-v). It appears that most but not all nitrotyrosine spots are associated with a TUNEL reaction (× 100). The control is negative (vi). (C) Nitrotyrosine, keratin, and iNOS triple staining. Epithelial cells (ii) are frequently iNOS⁺ (i), shown by the arrowhead, but keratin-negative cells are also iNOS⁺, shown by the arrow. Nitrotyrosine spots (iii) could include iNOS⁺ (iv) keratin-positive cells (v). Superimposition of antinitrotyrosine (iii) and iNOS (iv) staining is shown in panel Cvi. Superimposition of iNOS (iv) and antikeratin (v) staining is shown in panel Cvii. Superimposition of antinitrotyrosine (iii) and antikeratin (v) staining is shown in panel Cviii.