Journal: JCI insight

Article Title: Single-cell transcriptome analyses reveal microglia types associated with proliferative retinopathy.

doi: 10.1172/jci.insight.160940

Figure Lengend Snippet: Figure 6. The unique PRAM are localized near the pathological neovascular tufts. (A) Representative immunofluorescence images of PKM2 (green), microglia (IBa1, pink), vessel (isolectin B4, red), and nuclei (DAPI, blue) in whole-mount retinas of RA and OIR mice at P17. Scale bar: 50 μm (first, second, and fourth rows) and 10 μm (third and fifth rows). NC, negative controls; stained with isotype IgG of PKM2 and IBa1 antibody. The fluorescence intensity of PKM2 staining was calculated by ImageJ software (NIH) (5 images × 3 regions were obtained from n = 5 retinas of each group). Data are mean ± SEM. ***P < 0.001 versus RA by nonparametric Mann–Whitney U test. (B) FABP5 immunofluorescence staining of retinas from RA and OIR mice at P17. Rep- resentative FABP5 (green), CD11b (pink), isolectin B4 (red), DAPI (nuclei, blue), and merged images captured with confocal fluorescence microscopy. Scale bar: 50 μm (first, second, and fourth rows) and 10 μm (third and fifth rows). Stained with isotype IgG of FABP5 and CD11b antibody. The fluorescence intensity of FABP5 staining was calculated by ImageJ software (5 images × 3 regions were obtained from n = 5 retinas of each group). Data are mean ± SEM. ***P < 0.001 versus RA by nonparametric Mann–Whitney U test. (C) Representative immunofluorescence images of nuclear Ki67 (green), myeloid cells (CD11b, red), and vessel (isolectin B4, blue) in whole-mount retinas of RA and OIR mice at P17 (n = 4 per group). Scale bar: 50 μm and 20 μm (far right). (D) Immunofluorescence imaging of nuclear Ki67+ (white asterisk labelled, green), microglia/macrophages (F4/80, pink), and vessel (isolectin B4, blue) in whole-mount retinas of RA and OIR mice at P17 (n = 4 per group). Scale bar: 50 μm and 20 μm (far right).

Article Snippet: Samples were then incubated with primary antibodies against rabbit PKM2 (1:100, 4053, Cell Signaling Technology), rabbit Ki67 (1:200, RM-9106, Thermo Scientific), rabbit Fabp5 (1:200, 2.5 μg/mL, RD181060100, BioVendo), rabbit Igf1 (10 μg/mL, AF791, R&D Systems), rat F4/80 (1:100, ab6640, Abcam), rabbit IBa1 (1:400, 019-19741, Sakura Finetek), goat IBa1 (1:400, ab48004, Abcam), rat CD11b (5 μg/mL, 561114, BD Biosciences), and Alexa Fluor 488–, Alexa Fluor 594–, or Alexa Fluor 647–labeled Griffonia simplicifolia isolectin B4 (10 μg/mL; catalogs 121411, 121413, and I32450, respectively; Invitrogen) overnight at 4°C, followed by incubation with fluorescence-conjugated cross-adsorbed secondary antibody (1:500, Molecular Probes, Invitrogen) for 1 hour, and then counterstained with DAPI (Invitrogen).

Techniques: Immunofluorescence, Staining, Fluorescence, Software, MANN-WHITNEY, Microscopy, Imaging