confocal immunofluorescence microscopy  (Carl Zeiss)

Journal: Acta Neuropathologica Communications

Article Title: Divergent and convergent TMEM106B pathology in murine models of neurodegeneration and human disease

doi: 10.1186/s40478-025-02087-9

Figure Lengend Snippet: Comprehensive validation of a TMEM106B antibody using genetic and biochemical approaches. (A) Schematic of various commercial and non-commercial TMEM106B antibody epitopes. (B) The rabbit monoclonal TMEM106B antibody from Cell Signaling Technology (Catalog #93334, shown in green in the schematic) was found to be specific by Western blot analysis of wild-type (WT) and TMEM106B knockout (KO) mouse tissue. The antibody also showed specificity in mouse N2a WT versus KO cell lysate. Using another mouse astrocytic cell type, a gradient of siRNA treatment further demonstrated this specificity. Lastly, overexpression of the mouse TMEM106B sequence in HEK293 cells showed a dose-dependent immunoreactivity of the antibody. As the antibody epitope corresponds to residues surrounding alanine 17 of the TMEM106B sequence, the cleaved N-terminal fragment could also be detected . (C) Representative immunofluorescence images from TMEM106B WT and KO tissues stained with #93,334 (magenta) alongside neuronal marker NeuN (green) and nuclear marker (Hoechst). Scale bars = 5 μm

Article Snippet: Scale bar = 50 μm. (D) Immunofluorescence confocal microscopy for TMEM106B (Cell Signaling Technology #93334) does not reveal any overt differences in TMEM106B staining between non-transgenic and transgenic animals.

Techniques: Biomarker Discovery, Western Blot, Knock-Out, Over Expression, Sequencing, Immunofluorescence, Staining, Marker

Journal: Acta Neuropathologica Communications

Article Title: Divergent and convergent TMEM106B pathology in murine models of neurodegeneration and human disease

doi: 10.1186/s40478-025-02087-9

Figure Lengend Snippet: TMEM106B levels correlate with TDP-43 nuclear clearance in the motor cortex of (G 4 C 2 ) 149 mice. (A) Representative immunofluorescence images of the motor cortex and visual cortex from mice expressing either (G 4 C 2 ) 2 or (G 4 C 2 ) 149 repeats. Sections were stained for TMEM106B (red), TDP-43 (cyan), the neuronal marker NeuN (green), and nuclei (Hoechst, blue). Scale bars = 10 μm (B) Quantification of TMEM106B cytoplasmic intensity (left-hand side) and the TDP-43 nuclear-to-cytoplasmic (N/C) ratio (right-hand side) in the motor cortex (top) and visual cortex (bottom). No significant differences were observed between (G 4 C 2 ) 2 or (G 4 C 2 ) 149 groups in either region. Violin plots show individual cell data, while the overlayed data points represent the average per animal. A Mann-Whitney test was used to compare the average per-animal data points between groups. Motor Cortex TMEM106B intensity p = 0.5476 and Visual Cortex TMEM106B intensity p = 0.605, TDP-43 N/C Motor Cortex p = 0.3095, TDP-43 N/C Visual Cortex p > 0.9999. (C) Representative high magnification images of individual neurons in the motor cortex from (G 4 C 2 ) 2 and (G 4 C 2 ) 149 mice, showing nuclei with varying levels of TMEM106B expression. Dashed lines indicate nuclear boundaries. Scale bars = 5 μm. (D) Linear regression and correlation analyses between TMEM106B intensity and TDP-43 N/C ratio. A significant inverse correlation was observed in the motor cortex of (G 4 C 2 ) 149 mice (R 2 = 0.91, p = 0.0113), while no significant correlation was detected in the (G 4 C 2 ) 2 mice or in either group within the visual cortex. Each data point represents the average per-animal data

Article Snippet: Scale bar = 50 μm. (D) Immunofluorescence confocal microscopy for TMEM106B (Cell Signaling Technology #93334) does not reveal any overt differences in TMEM106B staining between non-transgenic and transgenic animals.

Techniques: Immunofluorescence, Expressing, Staining, Marker, MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: Divergent and convergent TMEM106B pathology in murine models of neurodegeneration and human disease

doi: 10.1186/s40478-025-02087-9

Figure Lengend Snippet: Cytoplasmic TMEM106B puncta are associated with decreased nuclear TDP-43 in the motor cortices of human C9-ALS and ALS/FTD cases. ( A ) Human motor cortex co-stained with TMEM-Sigma antibody, TDP-43 and NeuN. One neurologically healthy control, one C9-ALS patient (1) with severe TDP-43 nuclear clearance, and one C9-ALS patient (#2) with relatively intact TDP-43 localization are shown. Enlarged images of representative cells are outlined and shown in Figure ( D ). Scale bar = 20 μm. ( B ) Quantification of the percentage of neurons with intracellular TMEM106B puncta from healthy control ( n = 3), and C9-ALS and ALS/FTD ( n = 7) patients. Dots represent individual cases, bars represent means ± SEM. Mann-Whitney test, p = 0.9333. ( C ) Quantification of averaged TDP-43 nuclear to cytoplasmic (N/C) ratio from healthy control ( n = 3), and C9-ALS and ALS/FTD ( n = 7) patients. Dots represent individual cases, bars represent means ± SEM. Unpaired t-test, p = 0.9. ( D ) Zoomed-in images of individual cells showing an example neuron with TMEM106B cytoplasmic puncta (cell iii) with severe TDP-43 nuclear clearance. Scale bar = 5 μm. ( E ) Quantification of TDP-43 N/C ratio in neurons with TMEM106B cytoplasmic puncta (TMEM106B+) and those without (TMEM106B-) across all healthy controls, C9-ALS, and ALS/FTD ( n = 7) patients. 10 total cases for TMEM106B- ( n = 3 healthy control, n = 7 C9 patients) and 6 total cases for TMEM106B+ ( n = 2 healthy control, n = 4 C9 patients) are plotted, as not all cases showed TMEM106B cytoplasmic puncta ( B ). Violin plots show individual cell data, while the overlayed data points represent the average per case. A Mann-Whitney test was used to compare the average per-case data points between groups, p = 0.0934. ( F ) Quantification of the TDP-43 N/C ratio of neurons with or without TMEM106B cytoplasmic puncta from healthy controls or C9-ALS and ALS/FTD patients grouped by both patient diagnosis and TMEM106B phenotype. Violin plots show individual cell data, while the overlayed data points represent the average per case. A two-way ANOVA with multiple comparisons test was performed on the per-case data. *, p < 0.05

Article Snippet: Scale bar = 50 μm. (D) Immunofluorescence confocal microscopy for TMEM106B (Cell Signaling Technology #93334) does not reveal any overt differences in TMEM106B staining between non-transgenic and transgenic animals.

Techniques: Staining, Control, MANN-WHITNEY, Biomarker Discovery

Journal: Acta Neuropathologica Communications

Article Title: Divergent and convergent TMEM106B pathology in murine models of neurodegeneration and human disease

doi: 10.1186/s40478-025-02087-9

Figure Lengend Snippet: TMEM106B does not have altered distribution in mice expressing ALS-mutant G93A SOD1. (A) Midbrain/hindbrain region in non-transgenic (nTg) animals and animals expressing G93A SOD1 with DAB staining against SOD1. Vacuolization is apparent in transgenic animals. Scale bar = 25 μm. (B) Representative images of midbrain/hindbrain region in nTg and G93A SOD1 animals with DAB staining against Iba1 showing increased Iba1 reactivity in transgenic animals, as well as vacuolization. Scale bar = 25 μm. (C) Immunofluorescence confocal microscopy on the midbrain of either non-transgenic or SOD1 G93A transgenic animals shows clear overexpression of SOD1 in the transgenic animals and vacuolization. Images are representative of n = 8 nTg and 7 transgenic animals. Scale bar = 50 μm. (D) Immunofluorescence confocal microscopy for TMEM106B (Cell Signaling Technology #93334) does not reveal any overt differences in TMEM106B staining between non-transgenic and transgenic animals. Images are representative of n = 5 nTg and 5 transgenic animals. Scale bar = 50 μm. (E) Quantification of GFAP and TMEM106B within the hindbrain/midbrain region. Data points represent individual animals, bars represent means ± SEM. A Student’s unpaired t-test was performed, for GFAP p < 0.0001, and for TMEM106B p = 0.4730

Article Snippet: Scale bar = 50 μm. (D) Immunofluorescence confocal microscopy for TMEM106B (Cell Signaling Technology #93334) does not reveal any overt differences in TMEM106B staining between non-transgenic and transgenic animals.

Techniques: Expressing, Mutagenesis, Transgenic Assay, Staining, Immunofluorescence, Confocal Microscopy, Over Expression

Journal: Acta Neuropathologica Communications

Article Title: Divergent and convergent TMEM106B pathology in murine models of neurodegeneration and human disease

doi: 10.1186/s40478-025-02087-9

Figure Lengend Snippet: Correlation of TMEM106B and phosphorylated tau is observed in 12-month-old PS19 mice. (A) Representative images of CA1 and motor cortex with DAB staining against phosphorylated tau (AT8) in non-transgenic (nTg) animals and animals expressing the P301S mutant tau (PS19). Scale bar = 50 μm. (B) Immunofluorescence confocal microscopy of NeuN, TMEM106B (Cell Signaling Technology #93334) and AT8 in the dentate gyrus region of the hippocampus. Scale bar = 10 μm. PS19 animals that showed high AT8 signals tend to show higher TMEM106B signals. Quantification of AT8 (C) and TMEM106B (D) staining in 12-month-old mice shows that there is a significant difference in the AT8 signal intensity between transgenic and nTg mice. Bars represent means ± SEM. A student’s t-test was used to compare nTg and transgenic animals; (C) p = 0.0026, (D) p = 0.5785. (E) Linear regression and correlation analysis between TMEM106B and AT8 signals. (F) High magnification images from different PS19 animals showing limited co-localization between TMEM106B and AT8 signals. White lines indicate the linear regions of interest (ROIs) used for intensity profile analysis. (G) Corresponding fluorescence intensity profiles of TMEM106B (red) and AT8 (blue) along the ROIs marked in (F) for each cell. Plots show variable levels of spatial overlap and correlation between TMEM106B and AT8 signals across different cells

Article Snippet: Scale bar = 50 μm. (D) Immunofluorescence confocal microscopy for TMEM106B (Cell Signaling Technology #93334) does not reveal any overt differences in TMEM106B staining between non-transgenic and transgenic animals.

Techniques: Staining, Transgenic Assay, Expressing, Mutagenesis, Immunofluorescence, Confocal Microscopy, Fluorescence

Journal: Acta Neuropathologica Communications

Article Title: Divergent and convergent TMEM106B pathology in murine models of neurodegeneration and human disease

doi: 10.1186/s40478-025-02087-9

Figure Lengend Snippet: TMEM106B pathology does not correlate with tau pathology in Alzheimer’s disease (AD) and AD with limbic-predominant age-related TDP-43 encephalopathy (LATE). ( A ) Maximum projection images of human hippocampal tissue stained for nucleus (Hoechst), NeuN, phosphorylated Tau (pTau; AT8), and TMEM106B (SAB2106773, Sigma-Aldrich). Scale bar = 20 μm. ( B ) Quantification of AT8 staining normalized to NeuN staining for control, AD, and AD/LATE patients. Each data point represents one patient, and bars represent means ± SEM. At least 12 cells were counted per patient, with at least 95 cells counted in total for each group, n = 5. An ordinary one-way ANOVA ( p = 0.0021) with Tukey’s multiple comparisons test was used to compare each group. Control vs. AD p = 0.0196, control vs. AD/LATE p = 0.0012. ( C ) Quantification of TMEM106B staining normalized to NeuN staining for control, AD, and AD/LATE patients. Each data point represents a patient, bars represent means ± SEM. At least 12 cells were counted per patient, with a total of at least 95 cells counted for each group ( n = 5). An ordinary one-way ANOVA ( p = 0.3073) with Tukey’s multiple comparisons test was used to compare each group. Control vs. AD p = 0.8841, control vs. AD/LATE p = 0.2436. ( D ) A simple linear regression and a Pearson correlation coefficient of the intensity of AT8 and TMEM106B signal intensity for each patient. Each dot represents the AT8 and TMEM106B signal from the averaged cell value per patient; dots are colored by patient condition. At least 12 cells were counted per patient, with a total of at least 95 cells counted for each group ( n = 5). Line scans of intensity in patients with AD ( E ), or AD/LATE ( F ) indicate that the AT8 and TMEM106B signals are not colocalized. Scale bar = 5 μm. The highest signal for each channel was set to 1 and used to normalize all other values. Scans were performed over the white dashed line shown in the images for the individual channels

Article Snippet: Scale bar = 50 μm. (D) Immunofluorescence confocal microscopy for TMEM106B (Cell Signaling Technology #93334) does not reveal any overt differences in TMEM106B staining between non-transgenic and transgenic animals.

Techniques: Staining, Control

Journal: bioRxiv

Article Title: Integrating Cell Painting and Thermal Proteome Profiling for Inference of Targets and Mechanism of Action

doi: 10.1101/2025.05.30.657006

Figure Lengend Snippet: A.Lollipop chart for subcellular location of the 67 versus 40 proteins found to be either stabilized or destabilized in TPP for compounds (+)-JQ1 (top) and I-BET151 (middle), respectively. Subcellular location of each protein, based on immunohistochemistry and confocal microscopy, was retrieved from the Human Protein Atlas ( proteinatlas.org ). When a protein had more than one subcellular location assigned, that protein was included once per subcellular location in the graph. Bottom graph show a Venn diagram of the overlap between proteins stabilized/destabilized in (+)-JQ1 and I-BET151. Only 11 proteins were found to overlap between the two compounds. B. Physical protein-protein interaction (PPI) networks for the proteins found to be either stabilized or destabilized in TPP for compounds (+)-JQ1 and I-BET151, respectively. PPI networks were retrieved from the STRING db. Nodes are colored by subcellular location (from Human Protein Atlas). Large nodes correspond to proteins detected by TPP, where small nodes are additionally added nodes from the STRING db during network retrieval. C. Top: Physical PPI networks for (+)-JQ1 (same as B), except green nodes here indicate proteins found to be stabilized by (+)-JQ1 in cell extract. Bottom: Venn diagram for overlap of proteins detected in whole cells versus cell extract after perturbation with (+)-JQ1. D. Radar chart for features in cell painting data for (+)-JQ1 and I-BET151. Features were grouped into categories based on two criteria: (i) Cell Profiler module, i.e. Intensity (I), Correlation (C), Granularity (G), Location (L) and RadialDistribution (RD); and (ii) stains, i.e. Nucleus (Hoechst), ER (Concanavalin A), Nucleoli and cytoplasmic RNA (SYTO14), Golgi apparatus and F-actin cytoskeleton (WGA and Phalloidin) and Mitochondria (Mitotracker). Features were only consider for the object Cell, except for features from the stain for Nucleus, which was only considered in the object Nucleus. Additionally, area-shape related features were grouped by cell compartment, i.e. Cell (C), Cytoplasm (Cy) and Nucleus (N). E. t-SNE for the morphological features in the SPECS cell painting data on U2OS cells. Blue dots show the location of all cells treated with BET bromodomain inhibitors sharing at least one target with either (+)-JQ1 or I-BET151. Despite several BET bromodomain inhibitors clustering close to (+)-JQ1 or I-BET151, there are also several compounds with distinctly different morphological changes.

Article Snippet: Subcellular location for 17,025 proteins, based on immunofluorescence (ICC-IF) and confocal microscopy, was retrieved from the Human Protein Atlas database ( proteinatlas.org ) [ ] (retrieval date: 2024-10-29).

Techniques: Immunohistochemistry, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Integrating Cell Painting and Thermal Proteome Profiling for Inference of Targets and Mechanism of Action

doi: 10.1101/2025.05.30.657006

Figure Lengend Snippet: A.t-SNE for CP SPECS data showing (+)-JQ1 (yellow), I-BET151 (red) and a cluster of similar compounds (blue) identified using the consensus clustering algorithm SC3s on 13 principal components. k for the k -mean clustering algorithm was varied between 100 and 180. B. Physical PPI network for known protein targets of the compounds identified in the cluster in F. (+)-JQ1 and I-BET151 were blinded from the compound list before retrieving known targets. Network is coloured by the number of times a protein target is present within the cluster. Large nodes indicate proteins also detected in TPP for (+)-JQ1. C-D. Physical PPI network combining proteins identified in TPP and CP cluster, after applying a betweeness centrality calculation on the nodes from CP clustering. The number of CP supplied nodes to keep was chosen as the minimum number of nodes needed to maximize connection between the TPP identified proteins. Top: (+)-JQ1, bottom: I-BET151. The nodes are colored by ( C. ) subcellular location and ( D. ) number of times a protein target is present within the cluster in A.

Article Snippet: Subcellular location for 17,025 proteins, based on immunofluorescence (ICC-IF) and confocal microscopy, was retrieved from the Human Protein Atlas database ( proteinatlas.org ) [ ] (retrieval date: 2024-10-29).

Techniques: