Mouse anti brdu g3g4

Third instar larvae were dissected in cold phosphate-buffered saline (PBS) and fixed in 4% formaldehyde/PBS for 20 min at room temperature. They were then washed and permeabilized in PBT (0.2% Triton X-100 in PBS) for 30 min and blocked in BBT (0.3% BSA, 250 mM NaCl in PBT) for 1 h. Samples were incubated overnight at 4°C with primary antibody diluted in BBT, washed three times (15 min each) in BBT and incubated with secondary antibodies and DAPI (1 μg/ml) for 1.5 hour at room temperature. After three washes with PBT (15 min each), wing discs were placed in mounting medium (80% glycerol/PBS containing 0.05% n-Propyl-Gallate). All steps were performed on a rocking platform at the indicated temperature. The following primary antibodies were used: mouse anti-BrdU (G3G4; Developmental Studies Hybridoma Bank (DSHB)); mouse anti-MMP1 (3A6B4, DSHB); mouse anti-p53 (7A4, DSHB); rabbit anti-p-Histone H3 (sc-8656, Santa Cruz); rabbit anti-β-Gal (A11132, Invitrogen); and sheep anti-Digoxigenin-AP (#11093274910, Roche). The following secondary antibodies were used: anti-mouse IgG-Alexa Fluor 594; anti-mouse IgG-Alexa Fluor 488; anti-rabbit IgG-Alexa Fluor 594; and anti-rabbit IgG-Alexa Fluor 488 (Jackson InmunoResearch).

Three- to 7-d-old larvae were bathed in 10 mm BrdU or 3.3 mm EdU for 1–3 h, while adults were bathed for 2 d in 10 mm BrdU dissolved in 0.5% DMSO. Stocks solutions were 100 and 33.3 mm, respectively. Following fixation in 4% PFA BrdU was detected in tissue sections using the rat anti-BrdU (Abcam) or mouse anti-BrdU G3G4 (Developmental Studies Hybridoma Bank) antibodies at dilutions of 1:300 and 1:10, respectively. EdU was detected in fixed, dissected larval brains as described in the Click-It EdU Labeling kit (Invitrogen).