Localization of TIMP-1, gelatinase B, and TNF-α mRNA in the distal segment of injured nerve by in situ hybridization. Serial longitudinal cryosections of 4-d post-crush nerve were hybridized with digoxigenin-labeled antisense RNA probes to TIMP-1, gelatinase B, and TNF-α and counterstained with Hoechst 33258. (A) Hematoxylin-and-eosin staining of injured nerve. The enclosed areas in A, the crush site (*) and distal segment, are shown at higher magnification in B and D–S. D, H, L, and P, marked with * show the crush sites. E–G, I–K, M–O, and Q–S are distal segments. (B) Control showing the crush site hybridized with TIMP-1 sense probe. The black material at the top is the India ink marking the injury site. (C) Section of contralateral nerve hybridized with TIMP-1 antisense probe. (D) Crush and (E) distal segments hybridized with antisense TIMP-1 probe. (F) Hybridization with gelatinase B and (G) TNF-α antisense probes in the distal segment. (H–K) Hoechst nuclear staining (fluorescence) of sections in (D–G). (L–O) Corresponding staining for macrophages with mAb F4/80 on adjacent sections. (P–S) Corresponding staining for Schwann cells with S-100 antibody on adjacent sections. Thick arrows indicate cells expressing mRNA, and thin arrows indicate nonexpressing cells. Note that all macrophages express TIMP-1, gelatinase B, or TNF-α. Bars (A) 200 μm; (P) (for B–S), 15 μm.