software colocalization analysis  (MetaMorph Inc)

Journal: bioRxiv

Article Title: Characterization of the BRAF interactome identifies BRAF V600E <=>TP53 interaction in melanoma

doi: 10.1101/2025.06.20.660711

Figure Lengend Snippet: A. HEK293 cells (wild-type BRAF). B. Mel-9 cells (NRAS Q61R , wild-type BRAF). C. SKMEL-239 cells (heterozygous for BRAF V600E ). D. A375 cells (homozygous for BRAF V600E ). E. Quantification of colocalization of BRAF and TP53 in the cell lines listed above (quantification of n=4-5 fields of view from each of n=3 biological replicates; one-way ANOVA with multiple comparisons. **** = adjusted p-valued <0.0001). F. Quantification of TP53 localization in the nucleus by DAPI staining in the cell lines listed above (quantification of n=4-5 fields of view from each of n=3 biological replicates; one-way ANOVA with multiple comparisons. * = adjusted p-valued <0.03, *** = adjusted p-value <0.0009). Immunofluorescence microscopy depicting TP53 (magenta) and BRAF (green) localization in: G. Primary epidermal melanocytes expressing wild-type BRAF with no doxycycline. H. Primary epidermal melanocytes expressing BRAF V600E with the addition of doxycycline. I. Quantification of TP53 and BRAF colocalization in the epidermal melanocytes (quantification of n=4-5 fields of view from each of n=3 biological replicates; unpaired t-test p-value <0.0001). J. Quantification of TP53 localization in the nucleus by DAPI staining (quantification of n=4-5 fields of view from each of n=3 biological replicates; unpaired t-test p-value <0.0001). K. Immunoblots detecting BRAF and TP53 localization in the cytoplasm vs. nucleus after cell fractionation of SKMEL-239 cells, A375 cells, and HEK293 cells. L. Immunoblots detecting BRAF and TP53 localization in the cytoplasm vs. nucleus after cell fractionation of primary epidermal melanocytes with and without the addition of doxycycline to turn on BRAF V600E expression.

Article Snippet: Colocalization was quantified on a Lecia confocal microscope using the Leica LasX colocalization analysis (version 3.5.7.23225) software to quantify the overlap between fluorescent signals.

Techniques: Staining, Immunofluorescence, Microscopy, Expressing, Western Blot, Cell Fractionation

Journal: Brain Communications

Article Title: Chronic inflammation with microglia senescence at basal forebrain: impact on cholinergic deficit in Alzheimer’s brain haemodynamics

doi: 10.1093/braincomms/fcae204

Figure Lengend Snippet: Coexistence of tauopathy (hTau and pTau 181) in selected ChAT + cell clusters within BF of 3xTgAD mice . ( A ) Representative image showing mutant hTau (cyan) deposition in selected ChAT + cell cluster (magenta) at HDB on a sagittal section of a young 3xTgAD mouse (6 M). ( B ) Scatterplot of pixel intensities of cyan (quadrant 1), magenta (quadrant 2) and colocalization pixels (quadrant 3). ( C–D ) Higher magnification image showing colocalization of hTau and ChAT + at HDB and its colocalization pixels at quadrant 3 only on scatterplot. Pearson’s correlation coefficient (R p ) = 0.42 (ROI in circle). ( E–F ) Representative image on hTau (cyan) and ChAT + (magenta) expressing at BF from an old 3xTgAD mouse(18 M) and its scatterplot; R p = 0.20 on ROI. ( G ) Representative image on hTau deposition within ChAT + cell clusters at nBM and SI from a young 3xTgAD mice (9 M), R p = 0.19. ( H) Representative image on phosphor-tau Thr181 (cyan) deposition within ChAT + cell clusters at VDB in an old 3xTgAD mouse (20 M), R p = 0.22. ( I ) Average R p on colocalization of hTau and ChAT + cells at ROI within BF from multiple brain sections of young and old 3xTgAD mice (each dot represents one ROI). The average R p was higher in young versus in old AD mice. P = 0.004, unpaired 2-tailed t -test with equal variance, t = 3.205. df = 27; F = 1.476, P = 0.471. n = 3–5 mice/group. Abbreviations: R p : Pearson’s correlation coefficient. ROI: region of interesting. nBM: nucleus Basalis of Meynert; HDB: nucleus of horizontal limb diagonal band of Broca; VDB: nucleus of vert limb diagonal band. Scale bar, 200μm in panel A ; 100μm in panel C , E , G and H .

Article Snippet: For colocalization evaluation with double/triple immunohistochemistry staining on brain sections, Pearson’s correlation coefficient ( R p ) evaluation was applied using Zeiss confocal microscope (LSM710) ZEN software on ROI (region of interesting) from multiple images for statistical analysis (ZEISS: Acquiring and Analysing Data for Colocalization Experiments in AIM or ZEN Software).

Techniques: Mutagenesis, Expressing

Journal: Brain Communications

Article Title: Chronic inflammation with microglia senescence at basal forebrain: impact on cholinergic deficit in Alzheimer’s brain haemodynamics

doi: 10.1093/braincomms/fcae204

Figure Lengend Snippet: Iba1 + microglia interacting tauopathy with phenotypic changes in BF of 3xTgAD mice. ( A–B ) Representative images show Iba1 + microglia (magenta) at BF of an old WT mouse (2y) and scatterplot of colocalization pixels of Iba1(magenta) and hTau (cyan), which was almost undetectable in WT mice, R p = 0.03. ( C–D ) Representative images reveal Iba1 + microglia (magenta) were interacting hTau (cyan) within BF of a 3xTgAD mouse (14 M) and scatterplot on colocalization pixels at ROI. R p = 0.31. ( E ) Internalization, engulfing or phagocytosing hTau by Iba1 + microglia at BF of an old 3xTgAD mouse (2y). R p = 0.61. Microglia also showed heterogeneous morphology. ( F ) The average % of microglia associated with tauopathy (including phagocytosis) at BF was higher in 3xTgAD mice versus Age-matched WT mice. One-way ANOVA, F = 7.105, P = 0.005. Multiple comparison using unpaired 2-tailed t -test with equal variance between young WT and 3xTgAD mice, P = 0.013, t = 3.32, df =7; between old WT and 3xTgAD mice, P = 0.02, t = 3.5, df =5; n = 3–5 mice/group. ( G ) The average R p on colocalization of Iba1 and tauopathy at ROIs within BF from multiple brain sections of old 3xTgAD and WT mice, P < 0.0001, unpaired 2-tailed t -test with equal variance; t = 4.634, df =29. F = 1.567, P = 0.414. n = 3–5 mice/group. Scale bar, 100μm in panels 5A-a, c, e.

Article Snippet: For colocalization evaluation with double/triple immunohistochemistry staining on brain sections, Pearson’s correlation coefficient ( R p ) evaluation was applied using Zeiss confocal microscope (LSM710) ZEN software on ROI (region of interesting) from multiple images for statistical analysis (ZEISS: Acquiring and Analysing Data for Colocalization Experiments in AIM or ZEN Software).

Techniques: Comparison

Journal: Brain Communications

Article Title: Chronic inflammation with microglia senescence at basal forebrain: impact on cholinergic deficit in Alzheimer’s brain haemodynamics

doi: 10.1093/braincomms/fcae204

Figure Lengend Snippet: Increased expression of the senescence marker p16 INK4A in Iba1 + microglia and hTau harbouring cells with aging in BF of 3xTgAD mice . ( A–B ) Representative Immunofluorescence images revealed cell senescence marker p16 INK4A (cyan) expressed in Iba1 + (magenta) microglia within BF of aged 3xTgAD mice ( R p = 0.67), compared to aged WT mice ( R p = 0.21). ( C ) p16 INK4A expression (cyan) was also observed in hTau-bearing (magenta) cells within BF of 3xTgAD mice (R p = 0.37). ( D ) The Iba1 + microglia expressing p16 INK4A (%) were evaluated within four groups, one-way ANOVA: F = 18.99, P < 0.0001. It was significantly higher in old versus in younger AD mice, P = 0.0007, t = 5.755, df = 7, and higher in old AD versus old WT mice, P = 0.001, t = 5.117, df = 7; unpaired 2-tailed t -test with equal variance, The data points were mean on 3–4 brain sections/each mouse, n = 3–5 mice/group. ( E ) The average R p on colocalization of Iba1 with p16 INK4A (ROIs) from multiple sections of WT and 3xTgAD mice in two age groups was compared. One-way ANOVA analysis: F = 51.36, P < 0.001. The average R p was significantly higher in old AD versus in young AD mice, and in old AD versus old WT mice, but was not significantly different between young AD versus young WT mice as indicated. Tukey’s multiple comparisons test with adjusted values of P < 0.01 was indicated, df = 32; n = 3–4 mice/group. ( F ) The average R p on colocalization of p16 INK4A or TNF-α expression in hTau-bearing cells at BF of old AD mice. Scale bar, 50μm in panels A–C ; BF, Basal forebrain.

Article Snippet: For colocalization evaluation with double/triple immunohistochemistry staining on brain sections, Pearson’s correlation coefficient ( R p ) evaluation was applied using Zeiss confocal microscope (LSM710) ZEN software on ROI (region of interesting) from multiple images for statistical analysis (ZEISS: Acquiring and Analysing Data for Colocalization Experiments in AIM or ZEN Software).

Techniques: Expressing, Marker, Immunofluorescence

Journal: Brain Communications

Article Title: Chronic inflammation with microglia senescence at basal forebrain: impact on cholinergic deficit in Alzheimer’s brain haemodynamics

doi: 10.1093/braincomms/fcae204

Figure Lengend Snippet: Persistent increase of TNF-α in SASP microglia within BF in 3xTgAD mice. Representative images showing the pro-inflammatory cytokine TNF-ɑ expressed in Iba1 + microglia and corresponding scatterplots on colocalization pixel intensity within BF of WT ( A–B ) and 3xTgAD ( C–D ) mice. ( E) Representative image showed the persistent expression of TNF-ɑ in Iba1 + microglia from a very old 3xTgAD mouse (30 M), which spread to neighbouring cells and extracellular microenvironment (Fi–iv). ( F ) The Iba1 + microglia cells with TNF-α expression also displayed phenotype and deteriorating changes. (G ) The average R p on colocalization of Iba1 with TNF-α was significantly higher in old 3xTgAD versus in old WT mice as evaluated from multiple brain sections. P < 0.001, unpaired 2-tailed t -test with equal variance, t = 6.584, df = 48; F = 1.761, P = 0.168; n = 3–5 mice/group. ( H–I ) The representative images revealed the area (%) with TNF-ɑ distribution at BF of WT (Hi–ii) and 3xTgAD (Hiii–iv) mice (16–24 months) by Image J analyses, P = 0.004; 2-tailed t -test with equal variance, t = 3.73, df = 10; F = 4.607, P = 0.119; 2–3 sections/mouse, n = 4 mice/group. Scale bars, 50μm in A & C ; 20μm in E & F ; 100μm in H; R p , Pearson’s correlation coefficient.

Article Snippet: For colocalization evaluation with double/triple immunohistochemistry staining on brain sections, Pearson’s correlation coefficient ( R p ) evaluation was applied using Zeiss confocal microscope (LSM710) ZEN software on ROI (region of interesting) from multiple images for statistical analysis (ZEISS: Acquiring and Analysing Data for Colocalization Experiments in AIM or ZEN Software).

Techniques: Expressing