Goat anti rat alexa 488

Cellular proliferation was measured by nuclear incorporation of BrdU in the lungs of E18.5 embryos. BrdU (0.1 mg/g body mass) (B5002, Sigma Aldrich) was injected intraperitoneally into pregnant female mice at E18.5 and 2 h later the animals were anesthetized with isoflurane prior to euthanasia by cervical dislocation and removal of the embryos. Each embryo was weighted and processed for paraffin sections. Three-micrometer sections were deparaffinized and rehydrated, submitted to antigen retrieval treatment as described above, treated with 2N HCl for 45 min at 37 °C, neutralized with borate buffer (0.1 M pH 8.5 three times for 10 min each), washed with PBS and blocked with PBS, 0.2% Tween20, 5% BSA and 2% goat serum. After PBS washing, sections were immunostained overnight at 4 °C with a primary antibody for BrdU (1:2000, Accurate Chemical, OBT0030CX, diluted in PBS, 0.1% Tween20, 2% BSA and 2% goat serum). Sections were washed with PBS and incubated with the secondary antibody (1:500 diluted, goat anti-rat Alexa 488 from Jackson ImmunoResearch) and counterstained with nuclear marker DAPI (Sigma) for 1 h at RT. Preparations were then washed with PBS and mounted with ProLong Diamond anti-fading reagent (P36970, Life Technologies).

Differentiated rosette leaves from 4-week-old Arabidopsis thaliana (L.) Heynh. (Columbia) plants grown under short-day conditions (8h light/16h darkness) were fixed for 20min under vacuum in 4% formaldehyde in TRIS buffer (pH 7.5) and homogenized in TRIS buffer. Suspended nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg ml^–1) and flow-sorted according to their ploidy level using a FACS Aria flow cytometer (BD Bioscience) onto 22×22mm high precision coverslips (Marienfeld, Germany) in a drop of buffer containing 100mM TRIS, 50mM KCl, 2mM MgCl₂, 0.05% Tween, 5% sucrose, then air-dried and used for immunolabelling.
For co-localization and quantification of active and inactive modifications of RNAPII, immunostaining was performed according to Jasencakova et al. (2000) (link).
The non-phosphorylated (inactive) enzyme was detected with mouse monoclonal antibody (1:300; Abcam, ab817) and goat anti-mouse Alexa 488 (1:200; Invitrogen) or goat anti-mouse-Cy5 (1:300; Jackson ImmunoResearch).
RNAPIISer5ph (active; phosphorylated at Ser5) was detected with rabbit polyclonal antibody (1:200; Active Motif, 39233) and goat anti-rabbit Alexa488 (1:200; Jackson ImmunoResearch), and RNAPIISer2ph (active; phosphorylated at Ser2) with rat monoclonal antibody (1:200; Millipore, 04-1571) and goat anti-rat Alexa488 (1:200; Jackson ImmunoResearch).