Ab150092

Immunofluorescence specific for TNFα was performed on IVDs from the Transport study to investigate whether TNFα was able to penetrate the intact IVD. All samples were processed, embedded in plastic and sagittally sectioned (5μm) as previously described [33 (link)]. Prior to staining all samples were deplasticized. A primary polyclonal rabbit-anti human TNFα antibody (1:100 ab66579, Abcam Cambrdige, MA) and a goat anti-rabbit Alexafluor 594 secondary antibody (1:700 ab150092, Abcam) were used with omission of primary antibody as a negative control. All slides were counter stained with DAPI. The percentage of positively stained cells was calculated from each 20x image using ImageJ software for each region (annulus fibrosus: AF, 4 images; cartilage end-plates: CEP, 8 images; NP: 8 images).

The explants were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA, USA), frozen in liquid nitrogen, and sagittally sectioned (10 μm in thickness). Immunofluorescence (IF) was performed as previously described56 (link)57 (link). Briefly, the sections were post-fixed in 4% PFA for 40 min, washed with phosphate buffer saline (PBS) and 3% glycine, permealized with 0.03% sodium dodecyl sulfate, blocked with 3% goat serum, and then incubated with a primary antibody of rabbit anti-RFP (1:100, ab62341, abcam, Cambridge, MA, USA) at 4 °C overnight. After washes with PBS, the sections were incubated with an Alexa 594-conjugated goat anti-rabbit secondary antibody (1:1000, ab150092, abcam, Cambridge, MA, USA) for 2 h at RT. Then the sections were thoroughly washed and mounted with the ProLong Gold Antifade with DAPI reagent (Life Technologies, Grand Island, NY, USA). The images were taken under a fluorescence microscope (BX51, Olympus Optical Co. Ltd., Tokyo, Japan) as described above.