anti gfp d5 1 xp rabbit mab monoclonal antibody having cross reactivity  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish

doi: 10.1038/s41467-023-37026-6

Figure Lengend Snippet: a Strategy for the generation of Ara and Arb KI medaka strains. We designed a gRNA targeting ara intron 8 and arb intron 7 (the last intron). In the donor plasmids, we cloned a genomic fragment beginning from the end of exon 8 of ara and exon 7 of arb and ending just before the stop codon in the last exon, exon 9 of ara , and exon 8 (shown as black closed boxes with ‘E8’) of arb , where the 3xFLAG sequences (shown as blue boxes) and a P2A (2A peptide from porcine teschovirus-1)-mClover3 cassette (shown as green boxes) was placed in the frame. Thus, endogenous Ara and Arb were expressed as FLAG fusion proteins. Both AR-FLAG and P2A-mClover3 were expected to be expressed under the control of the endogenous Ar promoter. To generate KI medaka, sgRNA (for genome digestion in the final intron), donor plasmid, and Cas9 mRNA were co-injected into one-cell-stage medaka embryos. After injection, concurrent cleavage of the targeted genomic locus and the donor plasmid resulted in the integration of donor plasmid DNA containing 3xFLAG-T2A-mClover3 by non-homologous end joining (NHEJ). The scheme shows the forward integration of 3xFLAG-T2A-mClover3. b Expression of mClover3 in adult males of the two knock-in medaka strains, ara FLAG-2A-mClover3 (Ara-KI) and arb FLAG-2A-mClover3 (Arb-KI). White, grey, and red arrows indicate the regions adjacent to pectoral, dorsal, and anal fins, respectively (Ara-KI). White, grey, and red arrows indicate the pectoral, dorsal, and anal fins, respectively (Arb-KI). c Immunohistochemical detection of FLAG-tagged endogenous Ara and Arb in longitudinal sections of papillary processes of the anal fin (6 μm thickness). The merged images represent red fluorescence for immunostaining of FLAG (anti-DDDDK-tag mouse mAb monoclonal antibody), green fluorescence for immunostaining of mClover3 (anti-GFP D5.1XP rabbit mAb monoclonal antibody), and blue fluorescence for nuclear staining by DAPI. The medaka Arb-FLAG, but not Ara-FLAG, translocated into the nuclei of cells located in the distal tip of a bone nodule of papillary processes (marked by white arrows). d Representative micrographs of Masson/trichrome staining and immunohistochemical detection of FLAG-tagged endogenous Ara and Arb in adjacent sections of the urogenital region (8 μm thickness). Nuclear localisation of Ara-FLAG and Arb-FLAG was observed in the medulla ventral to the sperm duct, where ar DKO exhibited hyperplasia. e, f Representative micrographs showing immunohistochemical detection of FLAG-tagged endogenous Ara and Arb, and DAPI counterstaining in the brain (12 μm thickness). f shows a higher magnification of the POA. Nuclear localisation of both Ara-FLAG and Arb-FLAG was observed in the POA. n = 6 for Ara-KI and Arb-KI males, respectively.

Article Snippet: Briefly, after antigen retrieval by incubating slides in 0.1 mM citrate buffer (pH 6.0) in an autoclave (121 °C) for 1 min, the sections were blocked for 1 h using 1xPBS containing 0.1% Tween 20, 2% BSA, and 2% foetal bovine serum, and incubated with anti-DDDDK-tag (FLAG) mouse mAb monoclonal antibody (M185-3L, MBL, Nagoya, Japan, 1:300 dilution) and anti-GFP D5.1XP rabbit mAb monoclonal antibody having cross-reactivity to the mClover3 (#2956, Cell Signaling, Danvers, MA, USA, 1:300 dilution) overnight at 4 °C.

Techniques: Clone Assay, Plasmid Preparation, Injection, Non-Homologous End Joining, Expressing, Knock-In, Immunohistochemical staining, Fluorescence, Immunostaining, Staining