Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 1 | Multiplexed staining of various disease pathologies. A sequential multiplexed staining and analysis, known as QUIVER (Ref [34]), was employed on human FFPE tissue. The procedure started with the staining for IBA1 to assist in image deconvolution and merging. This step uti- lized a permanent chromogen to preserve the staining throughout each subsequent round. Subsequent staining rounds were performed for ITM2B (A and B), AT8 (C), and pTDP-43 (D), sequentially, using a removable chromogen. Post-deconvolution of single-channel IHC images, merged pseudo- fluorescent images were generated for each channel (i). Using HALO software (Indica Labs, version 3.6), a digital markup for each stain was also created (ii) to selectively detect each pathology. To focus on neuronal ITM2B structures (A), the algorithm was turned to omit large plaque-like structures over 1000 μm2 (Bii). Photos captured at 20× magnification. Image deconvolution and markup were completed in HALO software. Arrows denote pathological neuronal ITM2B staining.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Staining, Generated, Software

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 2 | Multiplexed staining of ITM2B co-localization in the human hippocampus. A method of sequential multiplexed staining and anal- ysis, known as QUIVER [34], was employed on 16 sections of human FFPE tissue. The procedure began with the staining for IBA1 followed by pT- DP-43 and AT8. ITM2B staining could be observed throughout the entirety of cells, including the apical dendrite co-localized with AT8 in ADNC cases (A). Co-localization was also seen frequently in pure LATE-NC (B). Additionally, cells positive for ITM2B, pTDP-43, and AT8 were seen in ADNC+LATE-NC cases (C). ITM2B-positive neurons that were also double-positive for AT8 and pTDP-43 showed several phenotypes. Their pres- ence was observed in high-intensity ITM2B stained neurons (C) in addition to low-intensity neurons more consistent with the staining pattern ob- served in other regions of the brain (D). Pseudofluorescent images were produced using the object co-localization algorithm in the HALO software. Scale bars = 50 μm. Image insets depict staining before deconvolution.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Staining, Produced, Software

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 3 | ITM2B immunostaining. Stained hippocampal sec- tions showed several ITM2B phenotypes in various disease states. Physiological ITM2B staining (low-pathology cases) demonstrated ro- bust immunoreactivity throughout the entire cell in nearly all regions of the hippocampus (A). However, pathological ITM2B could also be observed throughout the hippocampus. In ADNC cases, ITM2B with- in cells could show decreased cytoplasmic reactivity and pronounced puncta throughout the cell (B). Similarly, ITM2B also localized with plaque-like structures resembling compact dense plaques (C) or larg- er, more diffuse plaques (D). Photomicrographs captured at 20× mag- nification. Arrows indicate intraneuronal ITM2B immunoreactive structures. Arrowheads denote ITM2B immunoreactive plaque-like structures.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Immunostaining, Staining

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 4 | Digital analysis of ITM2B immunolabeling, stratifying by disease pathology. In addition to ITM2B, levels of AT8+ pTau (A) and pT- DP-43 (B) were quantified in patients with ADNC, LATE-NC, ADNC+LATE-NC, as well as normal control cases. Using the object co-localization algorithm in HALO software, we then quantified the total number of pTDP-43 inclusions that were also positive for AT8 (C).

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Immunolabeling, Control, Software

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 5 | Digital analysis of ITM2B, pTau, and pTDP-43 colabeling across a spectrum of pathologies. (A): Bar graph showing the density of ITM2B-positive cells per mm2 in different hippocampal subregions: Dentate gyrus (DG), CA3, CA2, CA1, and subiculum (Sub). Data are present- ed for control, ADNC (Alzheimer's disease neuropathologic changes), LATE-NC (Limbic-predominant age-related TDP-43 encephalopathy neuro- pathologic changes), AD+LATE-NC (co-occurrence of both ADNC and LATE-NC). (B): Percentage of ITM2B-positive cells co-localizing with AT8 (a marker for phosphorylated tau, indicating tauopathy). The inset (i) shows a correlation analysis between ITM2B and AT8 markers across all cases, with the linear regression line indicating a positive correlation. (C): Percentage of ITM2B-positive cells co-localizing with pTDP-43 (a marker for phosphorylated TDP-43, associated with LATE-NC). The inset (ii) shows a correlation analysis between ITM2B and pTDP-43 markers across all cas- es, with the linear regression line indicating a trend toward positive correlation.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Control, Marker

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 7 | ITM2B co-localization across a range of ADNC severity. Using sequential staining methods for ITM2B and Thioflavin-S, we ob- served several variations of ITM2B reactivity and association with AD pathology. Physiological intraneuronal ITM2B appeared to rarely co-localize with Thio-s (A), however, we also observed heavily punctated forms of ITM2B+ neuronal structures that co-localized with Thio-S+ fibrils, possibly representing a transition stage in the death of the cells (B). We also observed mature neurofibrillary tangles that were not co-localized with ITM2B (C). It is, therefore, possible that as Thio-S levels increase, the levels of ITM2B within a cell decrease, and mature tangles without ITM2B represent a neuron's end stage. Similarly, plaque-like structures could be observed in 3 general stages in diseased brains, including ITM2B+ without Thio-S (D), ITM2B+ with Thio-S (E) or Thio-S+ but ITM2B- (F).

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Staining

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 6 | Digital Pathological Markup showing ITM2B co-localization in disease-representative cases. After staining for ITM2B, pTDP-43 and AT8 (pTau), a digital markup representing each disease type was generated in HALO software to show the number of ITM2B+ cells also immunore- active for other markers. While ITM2B appeared to co-localize frequently with AT8 staining, it rarely colocalized with pTDP-43. In ADNC+LATE- NC brains, ITM2B occasionally co-localized with cells immunoreactive for both pTDP-43 and AT8. Each red dot represents a single pathological marker. Each black dot is a detected nuclei stained with hematoxylin.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Staining, Generated, Software, Marker

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 8 | Western blot analysis of ITM2B and PHF-1 across various subcellular fractions in samples from a normal control and an Alzheimer's disease case. The fractions analyzed include Low Salt (LS), Triton-X treated (TX), Sarcosyl (SARC), and detergent-insoluble, urea-soluble (Urea) fractions. Molecular weight markers are shown on the left. ITM2B (~40kDa expected MW) signals were enriched in the TX and SARC fractions, indicating membrane association, with in- creased PHF-1/pTau but not ITM2B levels in the Urea fraction of the Alzheimer's disease sample. β-Actin is used as a loading control.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Western Blot, Control, Molecular Weight, Membrane

Journal: iScience

Article Title: Divergent brain solute clearance in rat models of cerebral amyloid angiopathy and Alzheimer’s disease

doi: 10.1016/j.isci.2024.111463

Figure Lengend Snippet:

Article Snippet: Slides were incubated with mouse mAb CP-13 (1:200; a gift from Dr. Peter Davies, Litwin-Zucker Center for Research on Alzheimer’s disease at the Feinstein Institute) at 4°C overnight.

Techniques: Plasmid Preparation, Recombinant, Software