Rat anti rfp

For whole cell samples, DT40 cells were harvested, washed with PBS, and suspended in 1xLSB (Laemmli sample buffer) (final 1 × 10⁴ cells/μl), followed by sonication and heating for 5 min at 96 °C. Proteins were separated on SuperSep Ace, 5–20% (Wako) and transferred to Immobilon-P (Merck) using HorizeBLOT (ATTO). Primary antibodies used in this study were rabbit anti-chicken CENP-T (Hori et al. 2008 (link)), rabbit anti-chicken CENP-H (Fukagawa et al. 2001 (link)), rabbit anti-chicken Dsn1 (Hara et al. 2018 (link)), rabbit anti-GFP (MBL), rat anti-RFP (Chromotek), and mouse anti-α-tubulin (Sigma). Secondary antibodies were HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch), HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch), and HRP-conjugated anti-rat IgG (Jackson ImmunoResearch). To increase sensitivity and specificity, Signal Enhancer Hikari (Nacalai Tesque) was used for all antibodies. The antibodies were incubated with the blotted membranes for 1 h at room temperature or for overnight at 4 °C. Proteins reacting with antibodies were detected with ECL Prime (GE Healthcare) and visualized with ChemiDoc Touch (Bio-Rad). Acquired images were processed using Image Lab 5.2.1 (Bio-Rad) and Photoshop CC (Adobe).

Mice were anaesthetized with an overdose of sodium pentobarbital and transcardially perfused with saline, followed by 4% paraformaldehyde (PFA). Brains were post-fixed for 2 h at 4°C. Brains were sectioned on a sliding microtome or a vibratome at 60 μm. All primary and secondary antibodies were diluted in PBS containing 0.25% Triton X-100 and 2% BSA. The following antibodies were used rabbit anti-Calretinin (1:1000, Swant), chicken anti-GFP (1:3000, Aves Lab), goat anti-Prox1 (1:300, R&D systems), mouse anti-reelin (CR-50, 1:300, MBL international) and rat anti-RFP (1:500, ChromoTek). We used Alexa Fluor-conjugated secondary antibodies (Jackson ImmunoResearch Labs and Molecular Probes).

Third instar larvae were dissected in PBS and fixed with 4% paraformaldehyde, 0.1% deoxycholate (DOC) and 0.3% Triton X-100 in PBS for 27 min at room temperature. They were blocked in PBS, 1% BSA, and 0.3% Triton, incubated with the primary antibody over night at 4 °C, washed in PBS, 0.3% Triton and incubated with the corresponding fluorescent secondary antibodies for at least 2 h at room temperature in the dark. They were then washed and mounted in Vectashield mounting medium (Vector Laboratories).
The following primary antibodies were used: rat anti-Ci (DSHB 2A1) 1:50; mouse anti-Mmp1 (DSHB, a combination, 1:1:1, of 3B8D12, 3A6B4 and 5H7B11) 1:50; mouse anti-β-galactosidase (DSHB 40-1a) 1:50; rabbit anti-β-galactosidase (ICN Biomedicals) 1:2000; mouse anti-Wingless (DSHB 4D4) 1:50; rabbit anti-Dpp ([67 (link)]) 1:200; rabbit and mouse anti-PH3 (MERCK and Cell Signal Technology) 1:500, rat anti-RFP (Chromotek, 5F8) 1:2000.
Fluorescently labelled secondary antibodies (Molecular Probes Alexa-488, Alexa-555, Alexa-647, ThermoFisher Scientific) were used in a 1:200 dilution. Phalloidin TRITC (Sigma Aldrich) and Phalloidin-Alexa-647 (ThermoFisher Scientific) were used in a 1:200 dilution to label the actin cytoskeleton. TO-PRO3 (Invitrogen) and DAPI (MERCK) was used in a 1:500 dilution to label the nuclei.

We used chick anti-GFP (1:500, Abcam); rat anti-RFP (1:500, [5F8] Chromotek); mouse anti-nc82 (1:20, DSHB); and mouse anti-prospero (1:50, DSHB).

Embryo antibody staining and in situ hybridisation with exonic or intronic probes were as previously described [35 (link)]. The following primary antibodies were used: rabbit anti-Mef2 (1:800; from E. Furlong, EMBL, Germany), anti-β3-tubulin (1:5000; from R. Renkawitz-Pohl, Philipps Univ., Germany), anti-Lmd (1:1000; from E. Furlong), anti-GFP (1:500; Torrey Pines Biolabs), anti-RFP (1:1200; Rockland Immunochemicals), anti-LacZ CAPPEL (1:500; MP Biomedicals), anti-S59 (1:400; from M. Frasch, Erlangen, Germany), anti-Kr (1:500; from R. Pflanz, Goettingen, Germany), chicken anti-GFP (1:500; Abcam), mouse anti-Col (1:50), anti-FascII^ID4 (1:20; Hybridoma bank), and rat anti-RFP (1:500; Chromotek). Secondary antibodies were Alexa Fluor-488, -647 and -555 conjugated antibodies (1:300; Molecular Probes). DIG- or biotin-labelled RNA probes were transcribed in vitro using T7 or SP6 RNA polymerase, from PCR-amplified DNA sequences, either cloned by in pGemTeasy or directly. The sequence of primers used is given in Additional file 19: Supplementary Materials and Methods. Optimal confocal sections were acquired on Leica SP5, Leica SPE or Zeiss 710 microscopes at 40× magnification. Projections and 3D reconstructions were made using ImageJ and Volocity (PerkinElmer) software, respectively.