Alexa 488 anti rat

We stained free-floating coronal sections of the olfactory bulb from injected mice with primary antibodies: anti-rat Galectin-3 (1:300) and anti-rabbit Iba-1 (1:500, Wako/Nordic labs) with appropriate secondary antibodies Alexa-488 anti-rat, Alexa-647 anti-rabbit (raised in goat, 1:400, Invitrogen). We then analyzed these sections with a confocal laser microscope ZEISS LSM 510 (Switzerland), equipped with Ar and HeNe Lasers.

Primary antibodies used for Western blotting and immunofluorescence staining were mouse anti-K7 (RCK-105; Progen, Heidelberg, Germany), rat anti-K8 and rat anti-K19 (Troma I and Troma III, respectively; Developmental Studies Hybridoma Bank, Iowa, IA, USA), rabbit anti-K8 (273) and rabbit anti-K18 (275; kind gifts from J.E. Eriksson), rabbit anti-K20 (It-Ks 20.10; Epitomics, Burlingame, CA, USA), rat anti-Hsc70 (Enzo Life sciences; Farmingdale, NY, USA), mouse anti-K8 pS74 (LJ4; kind gift from M.B. Omary), rabbit anti-Ki67 (Abcam, Cambridge, MA, USA), rat anti-HSF2 (Abcam) and rabbit anti-IκB-α (Santa Cruz Biotechnology; Dallas, TX, USA). Secondary antibodies used for Western blotting were HRP-conjugated anti-mouse (GE healthcare, Little Chalfont, UK), anti-rat (GE healthcare and Cell Signaling Technology, Danvers, MA, USA) and anti-rabbit (Cell Signaling Technology) IgG antibodies. Secondary antibodies used for immunofluorescence staining were Alexa 488/Alexa 546 anti-mouse, Alexa 488 anti-rat and Alexa 488 anti-rabbit antibodies (Invitrogen, Carlsbad, CA, USA). Nuclei were stained with DRAQ5 (Cell Signaling Technology).

Fixed cells were rinsed with 0.1 M phosphate buffered saline (PBS) 3 times then blocked with 10 % normal donkey serum (NDS, Jackson ImmunoResearch, West Grove, PA) and 0.3 % Triton-X 100 (Sigma) in PBS for 30 min. Cells were incubated overnight in primary antibody, rat anti-BrdU (1:500, AbD Serotech, Raleigh NC) in 10 % NDS in PBS at 4 °C. Cells were then rinsed and incubated in secondary antibody, Alexa488 anti-rat (1:200, Invitrogen) in 10 % NDS in PBS. After rinsing, total BrdU+ cells were imaged and quantified using an automated Cellavista microscope system (Hoffman-La Roche, Basel, Switzerland).