Journal: bioRxiv

Article Title: Unconventional IFNω -like genes dominate the type I IFN locus and the constitutive antiviral responses in bats

doi: 10.1101/2024.02.22.581518

Figure Lengend Snippet: ( A ) The thermo-stability comparison. The specified IFN proteins (10 -3 µg/ml for each) were incubated at 4 ℃ or 40 ℃ for 4 hours, followed by incubation with RfKT cells at 37℃ for 6 hours. After a 24 hour infection, the expression levels of viral genes were analyzed using qPCR. The decreasing or increasing of V-RNA was shown as arrows. ( B ) Detection of the anti-proliferative activity of IFNs. Cell proliferation was measured by Cell Counting Kit-8 (CCK8) or BrdU assay measuring newly synthesized DNA in cells (0, 24, 48 or 72 h for CCK8, 48 h for BrdU). All experiments were repeated three times with similar results and a representative result is shown. One-way ANOVA with Dunnett’s correction was used for statistics (*P < 0.05; ***P < 0.001; ns no significance).

Article Snippet: BrdU that inserted to cell DNA was measured by anti-BrdU antibody (1:500, 66241-1-Ig, Proteintech) and a mouse anti-Cy3 antibody (1:100, ab6939, Abcam) to evaluate the cell proliferation rate.

Techniques: Comparison, Incubation, Infection, Expressing, Activity Assay, Cell Counting, BrdU Staining, Synthesized

Journal: bioRxiv

Article Title: Hedgehog signaling regulates neurogenesis in the larval and adult zebrafish hypothalamus

doi: 10.1101/740613

Figure Lengend Snippet: (A) Sagittal tissue section through the PR of a TgBAC(nes:EGFP) transgenic adult brain showing PCNA antibody labeled proliferative cells. Nestin expressing cells are predominantly PCNA negative. (B) Sagittal tissue section through the PR of a Tg(ptch2:EGFP) transgenic adult brain showing PCNA antibody labeled proliferative cells. Arrowheads indicate examples of PCNA labeled (proliferative) Hh-responsive cells. Panels at right show separated channels from the boxed region . (C) Hh-responsive cells (red) are largely distinct from Nestin expressing cells (green) in the posterior recess (PR) of the adult hypothalamus, as visualized in Tg(GBS-ptch2:NLS-mCherry;nes:EGFP) double transgenic fish. However, 17% of cells (74 cells of 442 total) contained both GFP and mCherry (arrowheads, n=9 tissue sections from 3 double transgenic fish) with GFP fluorescence substantially lower in double-labeled cells. Panels at right show separated channels from the boxed region. (D,E) BrdU pulse-chase experiment. Schematic timeline above panels shows timing of pulse and chase, with 47 dpf adult TgBAC(nes:EGFP) or Tg(GBS-ptch2:EGFP) fish being exposed to 10 µm BrdU in fish water for 2 days. 32 days later fish were sacrificed and tissue sections were labeled using anti-BrdU (red) and anti-PCNA (magenta) antibodies. ( D) Representative sagittal section through the posterior recess of a TgBAC(nes:EGFP) adult, insets show single channel data for the boxed region. A small number of Nestin-expressing cells in the posterior recess retained the BrdU label after one month. These cells did not express PCNA (arrowheads), indicating they were not in G1/S/G2 of the cell cycle at the time of fixation (n=3 fish, 13-16 tissue sections per fish). ( E) Representative sagittal section through the posterior recess of a Tg(GBS-ptch2:EGFP) adult (n=2 fish, 13-16 sections per fish), insets show single channel data for the boxed region. Most Hh-responsive cells failed to retain the BrdU label after one month and many of these cells expressed PCNA (arrowheads), indicating active cell cycling at the time of fixation. (F) Graph showing the percentage of Nestin-expressing or Hh-responsive cells that co-labeled with the BrdU or PCNA antibodies. (G) Quantification of BrdU label intensity in Nestin expressing and Hh-responsive cells showing BrdU labeling intensity was significantly lower in Hh-responsive cells compared to nestin expressing cells. *** p<0.001. **** p<0.0001. All panels show 0.5 µm single optical sections of 20µm tissue sections. Scale bars: 20µm.

Article Snippet: Following fixation in 4% PFA BrdU was detected in tissue sections using the rat anti-BrdU (Abcam) or mouse anti-BrdU G3G4 (Developmental Studies Hybridoma Bank) antibodies at dilutions of 1:300 and 1:10, respectively.

Techniques: Transgenic Assay, Labeling, Expressing, Fluorescence, Pulse Chase