Nl006

Mouse retina cryosections (10 μm) were fixed in 4% paraformaldehyde and blocked with 10% normal goat serum. The sections were rinsed with PBS, permeabilized with 0.1% Triton X-100, and incubated over night with anti-p38 antibody (rabbit polyclonal, ab7952, Abcam; Cambridge, MA). The slides were rinsed with PBS, and incubated with the anti-rabbit-FITC conjugated secondary antibody (NL006; R&D systems; Minneapolis, MN) for 1 hour [26 (link)]. After rinsing the slides with PBS, the sections were mounted with DAPI-containing mounting media (Vector Laboratories; Burlingame, CA), and imaged with an Olympus BX-UCB fluorescent microscope (20X magnification). The fluorescence intensity was quantified by using ImageJ software (version 1.44; NIH). Cryosections processed under similar conditions, except being incubated with the primary antibody, served as controls.

Spleen macrophages were sorted and stimulated with LPS for 1 h. Cells were then fixed in 4% paraformaldehyde (Sigma-Aldrich) and permeabilized with 0.1% triton X-100 (Sigma-Aldrich) at room temperature. Immunofluorescence staining was performed, as previously described [30 (link), 31 (link)]. Briefly, non-specific sites were blocked with 0.1% Tween-TBS with 5% BSA for 1 h at 37 °C. Incubations with primary antibodies were performed overnight at + 4 °C, and then cells were rinsed 3 times with 0.1% Tween-TBS. Incubations with secondary antibodies were performed for 1 h at 37 °C and then cells were rinsed 3 times with 0.1% Tween-TBS, stained with 4',6'-diamidino-2-phénylindole (DAPI) and mounted using Vectashield mounting medium (Vector Laboratories). Primary antibodies were directed against pP65 (cell signaling; 3033S), P65 (cell signaling; 6956P), pIKBα (cell signaling; 9246 s), pIKKβ (abcam; ab192440) and secondary antibodies were directed against donkey anti-mouse IgG Alexa Fluor 555-conjugated (Life technologies; A31571) and donkey anti-rabbit IgG NorthernLights493-conjugated (R&D systems; NL006) antibodies. Mean fluorescence intensity (MFI) was determined using image J software as previously described [32 (link)].