4′,6-diamidino-2-phenylindole (dapi) i counterstain (1,000 ng ml−1)  (Abbott Laboratories)

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Elevated TGFβ signaling contributes to ocular anterior segment dysgenesis in Col4a1 mutant mice

doi: 10.1016/j.matbio.2022.05.001

Figure Lengend Snippet: (A) qPCR analyses showing increased expression of the TGFβ target genes Ccn2, Cdkn1a, and Serpine1 in anterior segments from P7 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. (B and C) Schematic representation of the breeding strategy to generate Col4a1+/+;SBE-Luc and Col4a1+/Δex41;SBE-Luc reporter mice (B) and quantification of luciferase activity using an in vitro luciferase assay (C) showing a significant increase in TGFβ signaling in P7 anterior segments from Col4a1+/Δex41;SBE-Luc mice compared to their Col4a1+/+;SBE-Luc counterparts. n = 11 per genotype. Data shown as fold expression relative to Col4a1+/+;SBE-Luc mice. (D) Representative Western blot images (left) and quantification (right) showing increased ratio of phosphorylated to total SMAD2 (pSMAD:SMAD2) protein levels in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Because the pSMAD2 antibody recognized two bands, quantification was done separately for each band. (E) Representative Western blot images (left) and quantification (right) using antibodies recognizing both SMAD2 and SMAD3 showing increased pSMAD2:SMAD2 and pSMAD3:SMAD3 in P7 anterior segments from Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. n = 6 per genotype. Samples used in (D) and (E) were obtained from independent cohorts. (F) Representative images (left) of corneas immunolabeled for pSMAD2 (red) and counterstained with DAPI (blue), and quantification of corneal nuclear pSMAD2 labeling intensity (right) showing increased pSMAD2 levels in E18.5 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates. Scale bar = 20 μm. n = 7 Col4a1+/+ and 15 Col4a1+/Δex41 corneas. Data are presented as mean ± SD. *p < 0.05; **p < 0.01, Student’s t-test.

Article Snippet: Then, sections were washed in PBST and incubated in species-specific Alexa Fluor 488- or 594- conjugated secondary antibodies (ThermoFisher Scientific, 1:500) for 1 hr at room temperature, counterstained with DAPI (1 μg/ml), mounted in Prolong Gold Antifade Mountant (ThermoFisher Scientific) and imaged using a Zeiss LSM700 confocal microscope equipped with a Plan-Apochromat 63x/1.40 objective and ZEN software (Carl Zeiss Microscopy).

Techniques: Expressing, Luciferase, Activity Assay, In Vitro, Western Blot, Immunolabeling, Labeling

Journal: Cancer research

Article Title: Extracellular vesicle-derived miR-124 resolves radiation-induced brain injury

doi: 10.1158/0008-5472.CAN-20-1599

Figure Lengend Snippet: Representative images of Iba1+ microglia and CD68+ activated microglia immunohistochemistry are shown from the hippocampus and dentate gyrus (DG), respectively, for the 9 Gy miR-Scr and the 9 Gy miR-124 groups of the miR-124 cohort. Relative to miR-124-overexpressing group (B), the mice in the 9 Gy miR-Scr group show decreased numbers of Iba1+ cells in hippocampal subfields (A). miR-124 overexpression (D) resulted in a relative decrease in the CD68+ immunoreactivity in the DG region when compared to the 9 Gy miR-Scr group (C). Aggregate data from image processing with Imaris shows an increased Iba1-adjusted (E) volume of CD68 immunoreactivity in the irradiated group compared to the control and miR-124 overexpressing groups in the hippocampus (F). All data are presented as mean ± SEM (N = 4 mice per group). ** P < 0.01, *** P < 0.001 **** P < 0.0001 compared to the IRR group; P values are derived from ANOVA and Dunnett’s multiple comparisons test. Iba1, red; CD68, red; DAPI nuclear counterstain, blue. Scale bars = 150 μm (A, B) and 40 μm (C, D). dh, dentate hilus; gcl, granule cell layer.

Article Snippet: Tissues were then DAPI nuclear counterstained (1 μmol/L) and mounted using slow fade/antifade mounting medium (Life Technologies).

Techniques: Immunohistochemistry, Over Expression, Irradiation, Derivative Assay

Journal: Cancer research

Article Title: Extracellular vesicle-derived miR-124 resolves radiation-induced brain injury

doi: 10.1158/0008-5472.CAN-20-1599

Figure Lengend Snippet: Representative images of CD68+ activated microglia are shown from the dentate gyrus (DG) region of the hippocampus in all four groups for the five-week behavioral testing cohort. Relative to controls (A) the DG region of the hippocampus from irradiated mice show elevated levels of CD68 (B). EV treatment reduces CD68 levels in the irradiated brain (C and D; intracranial (IC) and retro-orbital (RO), respectively). Aggregate data from image processing with Imaris shows an increased volume of staining in the irradiated group compared to the control and EV-treated groups in the DG region in both the five-week (E) and six-month (G) cohorts. The same analysis showed similar trends in the CA1 region of the hippocampus in both the five-week (F) and six-month (H) cohorts. All data are presented as mean ± SEM (N = 4–6 mice per group). # P = 0.061, * P < 0.05, **** P < 0.0001 compared to the IRR group; P values are derived from ANOVA and Dunnett’s multiple comparisons test (all other groups compared to IRR group). CD68, red; DAPI nuclear counterstain, blue. Scale bars = 30 μm. dh, dentate hilus; gcl, granule cell layer

Article Snippet: Tissues were then DAPI nuclear counterstained (1 μmol/L) and mounted using slow fade/antifade mounting medium (Life Technologies).

Techniques: Irradiation, Staining, Derivative Assay

Journal: Cancer research

Article Title: Extracellular vesicle-derived miR-124 resolves radiation-induced brain injury

doi: 10.1158/0008-5472.CAN-20-1599

Figure Lengend Snippet: Fluorescently labeled hNSC-derived EV were transplanted using stereotactic intracranial (IC) or retro-orbital (RO) injections. Brain tissue was fixed at 48 hours post-surgery and brain sections were imaged using confocal microscopy. Analysis suggests that IC injected EV (A, C) and RO injected EV (B, D) are similarly effective in targeting the dentate gyrus (DG) (A, B) and CA1 (C, D) regions of the hippocampus. Fluorescently labeled EV membranes, red; DAPI nuclear counterstain, blue. Scale bars = 30 μm (retro-orbital method), 40 μm (intracranial method). dh, dentate hilus; gcl, granule cell layer; sr, striatum radiatum; pyr, pyramidal cell layer

Article Snippet: Tissues were then DAPI nuclear counterstained (1 μmol/L) and mounted using slow fade/antifade mounting medium (Life Technologies).

Techniques: Labeling, Derivative Assay, Confocal Microscopy, Injection