vectashield vibrance antifade mounting media with dapi  (Vector Laboratories)

Journal: Journal of Translational Autoimmunity

Article Title: T cell proliferative response to a homocitrullinated peptide correlates with joint pathology in collagen induced arthritis

doi: 10.1016/j.jtauto.2025.100345

Figure Lengend Snippet: Citrullinated and Homocitrullinated Proteins/Peptides are Present in DBA/1J Metatarsophalangeal Joints. A) Representative hematoxylin + eosin (H&E) at 80× magnification and immunofluorescent micrographs at 20× magnification of joints from DBA/1J mice immunized with bovine type II collagen (arthritic: N = 9; non-arthritic: N = 10) or PBS (N = 9). Scale bars represent 500 μm. Max projections of Z-stacks show nuclei (DAPI; blue), citrullinated proteins/peptides (CitP; green), and homocitrullinated proteins/peptides (HomoCitP; red). Corrected total fluorescence intensity for B) CitP and C) HomoCitP in three joint structures: bone marrow, synovium, and cartilage. Graphs show the median [IQR], with each symbol representing an individual mouse. Statistical analysis was performed using Kruskal-Wallis with Dunn's multiple comparisons test, and the resulting p-values were p = 0.0046, p = 0.0084, p = 0.4076, p = 0.0143, p = 0.0136, and p = 0.1733 respectively, with p-values for pairwise comparisons indicated on the graphs. ns: not significant.

Article Snippet: Following application of TrueView autofluorescence quencher, slides were mounted using Vectashield Vibrance Antifade mounting media with DAPI (SP-8500-15; Vector Laboratories; USA) and air dried for 2 h before imaging on the Nikon Ti2-E microscope.

Techniques: Fluorescence

Journal: Molecular cell

Article Title: Rate Limiting Enzymes in Nucleotide Metabolism Synchronize Nucleotide Biosynthesis and Chromatin Formation

doi: 10.1016/j.molcel.2025.11.009

Figure Lengend Snippet: (A) Schematic depicting the isolation of prenucleosomal and chromatin bound histones through Benzonase digestion (B) Immunoblot showing the total histones, and nascent and mature posttranslational modification marks associated with histones in prenucleosomal or chromatin fractions, with or without dTAG47 treatment for 24 h. Note: PRPS1 (HA) was detected only in pre-nucleosomal fraction, whereas H3K27ac was detected only in chromatin fraction. α-tubulin and H2A were used as loading controls for pre-nucleosomal and chromatin fraction. H3K9me3 and K27me3 were done in both pre-nucleosomal and chromatin fractions, whereas K36me3 and K79me3 were done only in chromatin fraction. (C) Schematic and experimental workflow showing histone-SNAP tag system to examine the new histone H3.3 and H4. ( D) Distribution of total (straight fixed) or chromatin bound (pre-extracted) histone H3.3 or H4 with or without PRPS1 depletion. Pseudo-colors were used in TMR-Star and DAPI panels to enhance contrast, scale bar: 5 μm . Violin plot below represents the quantification from n=3 independent experiments. p values were calculated from Student’s t-test. See Figure S2D – E for orthogonal projections showing TMR-Star and DAPI non-overlapping signal in straight fixed condition. (E) Schematic showing the workflow of isolation of proteins from nascent DNA. (F) iPOND demonstrating the histones H3, H4 and MCM2 associated with newly replicated DNA in multiple cell types with or without PRPS1 depletion (lysate). (G) Cell cycle phases distribution based on EdU-Alexa Fluor-647 and DAPI staining as measured through flow cytometry in multiple cell types treated with DMSO or dTAG47 for 24 h. Histogram is the representation of one of the three independent experiments and plots below represent the mean ± SD with individual values from three experiments. p values were computed from Student’s t test. Note: HCT116 PRPS1-HA-dTAG plot is also shown in Figure 6F for 0 and Day-1 time points. Representative histograms are separate experiments. (H) Fragment length distribution from nucleosome positioning analysis of ATAC-seq signal, with or without dTAG47 treatment for 24 hours. Fragment lengths (150–250 bp) corresponding to mononucleosome is highlighted. p value was computed using Wilcoxon-signed rank test, n =2 . Molecular weight (kDa) .

Article Snippet: Fixed cells were washed twice with 1x-PBS, mounted on VECTASHIELD antifade mounting media with DAPI (Vector Laboratories H-1800–2) and imaged on Zeiss LSM800 confocal microscope.

Techniques: Isolation, Western Blot, Modification, Staining, Flow Cytometry, Molecular Weight

Journal: Molecular cell

Article Title: Rate Limiting Enzymes in Nucleotide Metabolism Synchronize Nucleotide Biosynthesis and Chromatin Formation

doi: 10.1016/j.molcel.2025.11.009

Figure Lengend Snippet: (A-B) Schematic showing the degradation of PRPS1-HA-dTAG (A) or NASP-FLAG-dTAG (B) by dTAG47 (top) . Histone H3 and H4 levels in response to short and long-term depletion of PRPS1 (A) or NASP (B) . Respective plots below are the mean band intensities (± SEM) of H3 or H4 normalized to loading control from at least three experiments. (C) Fragment length distribution from nucleosome positioning analysis of ATAC-seq signal from WT or tNASP KO HEK293T cells. Fragment lengths (150–250 bp) corresponding to mononucleosome is highlighted. p value was computed using Wilcoxon-signed rank test. (D) Comparison of changes in nucleosome positioning resulting from PRPS1 or tNASP loss analyzed by computing the difference in fragment length distribution of dTAG47 treated versus tNASP KO samples to their respective controls. (E) Nucleosome positioning correlation with 95% confidence interval between PRPS1 depletion and tNASP KO analyzed by computing the difference in fragment length around mononucleosome (150–250 bps) of dTAG47 treated or tNASP KO samples to their respective controls. Pearson correlation coefficient, R 2 = 0.81. (F) Cell cycle phases distribution based on EdU-Alexa Fluor-647 and DAPI staining as measured through flow cytometry in HCT116 PRPS1-HA-dTAG treated with DMSO, dTAG47 for 1 or 7 days. Histogram is the representation of one of the three independent experiments and associated plots represent the mean ± SD with individual values from three experiments. p values were computed from Student’s t test. Note: Day-0 and Day-1 time points are also shown in Figure 3G for HCT116 PRPS1-HA-dTAG . Representative histograms are separate experiments. (G) Proliferation of HCT116 PRPS1-HA-dTAG cells over time, with or without PRPS1 depletion, as measured by IncuCyte imaging system. n=6, p value is from two-way ANOVA. Molecular weight (kDa) .

Article Snippet: Fixed cells were washed twice with 1x-PBS, mounted on VECTASHIELD antifade mounting media with DAPI (Vector Laboratories H-1800–2) and imaged on Zeiss LSM800 confocal microscope.

Techniques: Control, Comparison, Staining, Flow Cytometry, Imaging, Molecular Weight