Fig 1: Generation and evaluation of an Olfm4eGFP reporter mouse strain. (a) Schematic representation of the mouse Olfm4 locus (chr14: 79,998,501–80,000,501) target vector. The region was designed such that the long-homology arm (LA) extends ~ 5.04 kb 5' to the GFP sequence, and the short-homology arm (SA) extends 2.03 kb 3' to the FRT-flanked Neo cassette. The eGFP-2A coding sequence is fused right after the endogenous ATG initiation site in exon 1. The FRT-flanked Neo cassette is inserted 397 bp downstream of exon 1. MA, middle-homology arm; WT, wild-type. (b) PCR analysis for identifying recombinant expanded surviving clones of BA1 (129/SvEv x C57BL/6) ES cells after selection with G418 antibiotic. PCR product size: 2.21 kb. An x denotes expanded clones. DNA from an individual clone (before reconfirmation) was used as a positive control (+). No DNA (-) and wild-type DNA (WT) were used as negative controls. (c) Southern-blotting analysis for confirming correct homologous recombination in positive clones identified by PCR. The expected sizes for the wild-type (WT) allele are 22.5 kb and 7.4 kb; the expected sizes for the recombined (KI) allele are 10.499 and 13.062 kb. HYB, DNA from C57BL/6 (B6), 129/SvEv (129), and BA1 (129/SvEv x C57BL/6) (Hybrid) mouse strains were used as wild-type controls. ITL Ladder, 4–20 kb. (d) Representative PCR genotyping of wild-type, heterozygous, and homozygous mice. Wild-type allele = 289 bp, knock-in allele = 426 bp. M 100Bp, 100-bp ladder. (e–g) Representative single-color or merged images from double-color immunofluorescent staining in small-intestine tissues of P1 (e), P14 (f), or P56 (g) Olfm4eGFP mice with Olfm4 and GFP (ab13970, Abcam) antibodies. Scale bars: 20 µm. Blue represents DAPI nuclear staining. (h) Representative merged images from triple-color immunofluorescent staining in small-intestine tissues of P14 or P42 Olfm4eGFP mice with Olfm4, Foxa1, and GFP (top panel), Olfm4, Ck8, and Ck5 (middle panel), or Olfm4, Ck4, and Ck5 (bottom panel) antibodies. Scale bars: 20 µm. Blue represents DAPI nuclear staining.
Fig 2: Olfm4/eGFP is expressed during early postnatal development of Olfm4eGFP mouse prostate and urethral epithelium. (a, b) Representative merged and single-color images from triple-color immunofluorescent staining in P1 (a) or P7 (b) prostate tissues of Olfm4eGFP mice with Olfm4, E-cadherin, and GFP (ab13970, Abcam) antibodies (left panels) or with Olfm4, Foxa1, and GFP (ab13970, Abcam) antibodies (right panels). Ecd, E-cadherin. Scale bars: 20 µm. Blue represents DAPI nuclear staining. # indicates the region shown in the single-color staining micrographs. (c, d) Representative merged images from triple-color immunofluorescent staining in P14 (c) or P20 (d) urethral-tube or prostate tissues of Olfm4eGFP mice with Olfm4, Foxa1, and GFP (ab13970, Abcam) antibodies. UT, urethral tubes; AP, anterior prostate; DLP, dorsolateral prostate; VP, ventral prostate. Scale bars: 20 µm. Blue represents DAPI nuclear staining.
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