Journal: Nature medicine

Article Title: Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease.

doi: 10.1038/nm.3901

Figure Lengend Snippet: Figure 2 Renal epithelial cells undergo a partial EMT after UUO. (a) Ksp1.3-Cre;tdTomatofl/fl mice were subjected to UUO and kidneys were collected 15 ds after surgery. Representative images (n = 20) of sections of kidneys from Ksp1.3-Cre;tdTomatofl/fl mice (KCT) 15 d after being subjected to UUO. Images are for obstructed (KCT + UUO) and contralateral non-obstructed kidneys (Control) from KCT mice stained for LTA (Lotus tetragonolobus agglutinin) + PNA (peanut agglutinin) (markers of proximal and distal tubules17), α-SMA and DAPI. tdTomato fluorescent protein expression was directly visualized. Asterisks (*) indicate α-SMA expression in tubular cells that have lost lectin expression 15 d after UUO. dt, dilated tubule; g, glomerulus; t, tubule. (b) Number of dilated and total tubules in kidneys from WT and SFKC mice 15 d after UUO. Ten different fields were counted in 12 mice per group. Data represent mean ± s.e.m. *P < 0.05, NS, nonsignificant; Mann-Whitney test. (c) Representative images (n = 5) of double Snail1 and α-SMA immunolabeling in sections from obstructed kidneys of WT mice euthanized 15 d after surgery. White asterisks indicate Snail1-positive nuclei. Green asterisks indicate double-positive Snail1 and α-SMA tubular cells. (d) Representative images (n = 5) of lectin (LTA) and Snail1 immunofluorescence in sections from the same kidneys used in b. Scale bars, 50 µm.

Article Snippet: The sections were incubated overnight at 4 °C with the following primary antibodies: biotinylated Lotus tetragonolobus agglutinin, LTA (B-1325, Vector Laboratories, Burlingame, CA); biotinylated peanut agglutinin, PNA (B-1075, Vector Laboratories, Burlingame, CA); α smooth muscle actin, (α-SMA) (Sigma-Aldrich, St Louis, MO); vimentin (sc-7557, Santa Cruz Biotechnology, Santa Cruz, CA); Snail1 (Ab1773; Abcam PLC, Cambridge, MA); phosphoNF-κB p65 (Ser276) (3037S, Cell Signaling Technology, Inc., Danvers, MA); phospho-Smad2 (Ser465/467) (3101S, Cell Signaling Technology, Inc., Danvers, MA); F4/80 (MCA497GA, Serotec, Oxford,UK); estrogen receptor alpha, ER-α (sc-543, Santa Cruz Biotechnology, Santa Cruz, CA); CD163 (sc-18796 Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Control, Staining, Expressing, MANN-WHITNEY, Immunolabeling, Immunofluorescence

Journal: Data in Brief

Article Title: Imaging data on characterization of retinal autofluorescent lesions in a mouse model of juvenile neuronal ceroid lipofuscinosis (CLN3 disease)

doi: 10.1016/j.dib.2020.106076

Figure Lengend Snippet: Autofluorescent lesions in the homozygous Cln3 Δex7/8 mouse retinal OPL/INL were negative for the cone photoreceptor marker peanut agglutinin. Peanut agglutinin (PNA), a lectin protein derived from the fruits of Arachis hypogaea and with high affinity for galactose-galactosamine disaccharide residues, stains cone photoreceptors [ , ]. Hypopigmented homozygous Cln3 Δex7/8 mice (10-17 month old) on an outbred 129Sv/Ev/CD1 mixed background, which is known to harbor additional CLN3-independent retinal degenerative genetic loci, were shown to have significantly decreased cone photoreceptors by PNA chromogenic IHC [ , ]. However, we did not observe overt photoreceptor loss by Cy5-PNA fluorescence in a homozygous Cln3 Δex7/8 mouse (22 month old) on a C57BL/6J background. In addition, autofluorescent lesions in the homozygous Cln3 Δex7/8 mouse retinal OPL/INL were negative for Cy5-PNA. Autofluorescence was collected with a 487.9 nm laser and a 525/25 nm emission filter. PNA IF was performed on retinal cyro-sections stained with Cy5-conjugated PNA (Vector CL-1075a) and confocal fluorescent images were acquired with a 638.6 nm laser and a 700/75 nm emission filter. (A) Representative DIC and confocal fluorescent images of homozygous Cln3 Δex7/8 mouse retina show autofluorescent lesions in the OPL/INL (green channel; pointed by yellow arrows) and Cy5-PNA fluorescence (red; pseudo-color) at the cone IS and OS and cone pedicles in the OPL/INL. Note that DIC images clearly show distinctive morphologies of RPE, OS, IS, ONL, OPL, INL, IPL, and autofluorescent lesions. (B) Representative DIC and confocal fluorescent images of WT mouse retina show autofluorescence (green) and Cy5-PNA fluorescence (red; pseudo-color). (C) Control fluorescent images of homozygous Cln3 Δex7/8 mouse retina acquired and processed exactly as in (A) except that Cy5-PNA staining was omitted. Note that bringing up signals in the pseudo-red channel revealed autofluorescent lesions rather than the typical Cy5-PNA staining patterns (A-B) in the OPL/INL.

Article Snippet: PNA IF was performed on retinal cyro-sections stained with Cy5-conjugated PNA (Vector CL-1075a) and confocal fluorescent images were acquired with a 638.6 nm laser and a 700/75 nm emission filter. (A) Representative DIC and confocal fluorescent images of homozygous Cln3 Δex7/8 mouse retina show autofluorescent lesions in the OPL/INL (green channel; pointed by yellow arrows) and Cy5-PNA fluorescence (red; pseudo-color) at the cone IS and OS and cone pedicles in the OPL/INL.

Techniques: Marker, Derivative Assay, Fluorescence, Staining, Plasmid Preparation