n a trueview autofluorescence quenching kit vector laboratories sp 8400 15 vectashield vibrance antifade mounting medium  (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: Nature Communications

Article Title: Chimeras co-targeting antigens and FcγRIIb trigger degradation of extracellular soluble proteins and pathological aggregates

doi: 10.1038/s41467-025-67207-4

Figure Lengend Snippet: a Single-cell analysis reveals expression of FcγRIIb by various cell types in mouse brain. b Schematic diagram illustrating the structure of Lecanemab SK3 (Lec-SK3) and Lecanemab-N297A (Lec). c Schematic diagram illustrating how Lec-SK3 enhances FcγRIIb-mediated internalization of Aβ oligomers. d Representative confocal images showing Aβ oligomers (green) internalization in microglia treated with unlabeled Lec-SK3 (upper) or Cy5-labeled Lec-SK3 (lower). Cell nuclei were stained with DAPI (blue). Scale bar: 2 μm. Experiments were performed in triplicate and repeated three times with similar results. e Experimental timeline of 2.5-month-old male WT or 5xFAD mice with bilateral i.c.v. injection of saline, 2 mg/kg Lec-SK3 or Lec. Spontaneous alternation rate ( f ), exploration frequency and dwelling duration in the novel arm ( g ) of the Y-maze were assessed post-injection of saline, Lec-SK3 or Lec ( n = 7 mice per group). h Discrimination index (%) and recognition index (%) of the NOR test were determined following the injection of saline, Lec-SK3, or Lec ( n = 7 mice per group). i Representative confocal z-stack images (left) and 3D reconstructed images (right) of microglia (IBA1, green) and Aβ plaque (6E10, red) in CA1 regions of hippocampus from mice. Scale bar, 5 μm. Experiments were performed in triplicate and repeated three times with similar results. j Representative confocal images and quantifications of microglia (IBA1, green) and Aβ plaque (6E10, red) in CA1 regions of hippocampus from mice ( n = 9 independent fields from 3 mice per group). Cell nuclei were labeled with DAPI (blue). Scale bar, 100 μm. k Western blotting and comparisons of MAP2 and PSD95 in hippocampus of 5xFAD mice with different treatments ( n = 4 mice per group). Data are presented as means ± SEM. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, the exact P value were provided in Supplementary Table ). Panel ( b ) was created in BioRender. Zhao, J. (2025) https://BioRender.com/kr4x49j . Panel ( c ) was created in BioRender. Zhao, J. (2025) https://BioRender.com/6beshm0 . Panel ( k ) was Created in BioRender. Zhao, J. (2025) https://BioRender.com/l2up9v3 .

Article Snippet: The sections were washed with TBST (0.1% Tween 20 in 1 × TBS), and Vectashield with DAPI (Vector Lab, SP-8500-15) was used as mounting medium.

Techniques: Single-cell Analysis, Expressing, Labeling, Staining, Injection, Saline, Western Blot

Journal: Nature Communications

Article Title: Chimeras co-targeting antigens and FcγRIIb trigger degradation of extracellular soluble proteins and pathological aggregates

doi: 10.1038/s41467-025-67207-4

Figure Lengend Snippet: a Experimental timeline for 2-month-old male WT or 5×FAD mice receiving bilateral i.v. injections of saline, AAV-Lec-SK3, AAV-Lec, or AAV-GFP. b , c , Spontaneous alternation rate ( b ) and discrimination index of the NOR test ( c ) were assessed 60 days post-injection of saline, AAV-Lec-SK3, AAV-Lec, or AAV-GFP ( n = 6 mice per group). d Representative confocal images (left panel) and 3D-reconstructed images (right panel) of microglia (green) and Aβ plaques (red) in the CA1 region of the hippocampus at 60 days post-treatment. Scale bar: 5 μm. e Representative confocal images and quantification of microglia (green) and Aβ plaques (red) in the CA1 region of the hippocampus from mice 60 days post-treatment. Cell nuclei were stained with DAPI (blue). Scale bar: 50 μm. Quantification of Aβ and IBA1 levels using 9 independent fields from 3 mice per group. f Representative Western blot analysis of MAP2 and PSD95 in mice hippocampus 60 days post-treatment. g Quantification of MAP2 and PSD95 in panel ( f ) ( n = 5 mice per group). Data are presented as means ± SEM. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, the exact P value were provided in Supplementary Table ). Panel ( a ) was created in BioRender. Zhao, J. (2025) https://BioRender.com/53ug670 , https://BioRender.com/s49qd4y . Panel ( f ) was Created in BioRender. Zhao, J. (2025) https://BioRender.com/l2up9v3 .

Article Snippet: The sections were washed with TBST (0.1% Tween 20 in 1 × TBS), and Vectashield with DAPI (Vector Lab, SP-8500-15) was used as mounting medium.

Techniques: Saline, Injection, Staining, Western Blot

Journal: Nature Communications

Article Title: Chimeras co-targeting antigens and FcγRIIb trigger degradation of extracellular soluble proteins and pathological aggregates

doi: 10.1038/s41467-025-67207-4

Figure Lengend Snippet: a , b Effect of inhibitors targeting different endocytic pathways on FcRTAC internalization. Huvec-2b cells were pre-treated with endocytosis inhibitors for 1 h, followed by the addition of pH-sensitive reagent-prelabeled NK-2-12-SK3 ( a ) or NK-2-12-SK4 ( b ). Fluorescence changes were monitored hourly over 12 h. Chlorpromazine: clathrin-mediated endocytosis inhibitor; cytochalasin D: macropinocytosis and phagocytosis inhibitor; amiloride (EIPA): macropinocytosis inhibitor; nystatin: caveolae pathway inhibitor. c – e , Live-cell imaging of FcRTAC-mediated IgE endocytosis. Cy3-labeled IgE (red) was incubated with Huvec-2b cells in the presence or absence of Cy5-labeled FcRTACs (white) for 2 h. Cells were stained with LysoTracker (green) and Hoechst (blue) for confocal imaging. Images were acquired using a 40× objective on a confocal microscope ( c , scale bar: 20 μm) or a super-resolution microscope ( d and e ; scale bar: 10 μm). f Subcellular distribution of FcγRIIb and FcRTACs in Huvec-2b cells. Huvec-2b cells were incubated with Cy5-labeled FcRTACs (red) for 1 h. After fixation and permeabilization, cells were stained for Lamp-1 (green) and FcγRIIb (white) using primary antibodies and their corresponding fluorescent secondary antibodies. Nuclei were stained by DAPI (blue). Images were acquired using a 40× objective on a confocal microscope. Scale bar: 10 μm. c–f Experiments were performed in triplicate and repeated three times with similar results.

Article Snippet: The sections were washed with TBST (0.1% Tween 20 in 1 × TBS), and Vectashield with DAPI (Vector Lab, SP-8500-15) was used as mounting medium.

Techniques: Fluorescence, Live Cell Imaging, Labeling, Incubation, Staining, Imaging, Microscopy, Super-Resolution Microscopy