western blot quantification analysis used image lab software  (Bio-Rad)

Journal: bioRxiv

Article Title: Galectin-3 deletion modulates microglial phenotype and Aβ response via TREM2 activation while attenuating neuroinflammation

doi: 10.1101/2025.03.17.643790

Figure Lengend Snippet: A Aβ42 fibril formation monitored as a function of time through Thioflavin-T (ThT) fluorescence intensity in the presence of varying concentrations Gal3 (n=8/group). The kinetic traces were alternately fitted using the secondary nucleation-dominated model of AmyloFit , allowing the product of k + k 2 to vary freely (elongation and secondary nucleation, left) and k n k + (primary nucleation and elongation, right), while the other product was kept as global constant at the value obtained for Aβ alone. The best fit was obtained for k + k 2 , represented with a green tick (left). B SPR sensograms, response as a function of time, reporting on the interaction of Gal3 with immobilized mAβ (gray), and fAβ (orange). Solid lines show fits to the data during association (left), and dissociation (right). The data has been background subtracted and is represented as the average of two measurements. Time zero is set at the beginning of the injection (left) and after the injection (right). C Native PAGE Western Blot anti-Aβ (6E10) of different Aβ fibrillations over time with and without Gal3 protein. Timepoints were selected based on a ThT assay which was run before starting the incubations (Figure S2B). D SDS-PAGE Western Blot anti-Aβ (6E10) of different Aβ fibrillations over time with and without Gal3 protein. Same timepoints as in were applied (based on Figure S2B). E Representative TEM images of Aβ after fibrillation with and without Gal3 (first two images) plus Gal3 added only after fibrillation (third image). F Representative Cryo-EM images and quantification of fibril thickness (n≥12/group) of Aβ after fibrillation with and without Gal3 (first two images) plus Gal3 added only after fibrillation (third image). G Gold-immunolabeled electron micrographs of Gal3 in the hippocampus of APP/PS1 mice. A) Immunogold particles specifically labelled microglial cells (Microglia) in the vicinity of amyloid plaques (Plaque). Immunoelectron microscopy confirmed Gal3-labeling also in the extracellular space and in fibrillar aggregates. B) Higher magnification image showing gold particles within the microglial cytoplasm as well as extracellular (arrow). C) Detail image of Gal3 immunolabeling associated with extracellular bundles of plaque-associated amyloid fibrils. In A and B, values are expressed as mean ±SD. In F, each value represents an individual fibril thickness and mean ±SEM is represented. One-way ANOVA with Tukey’s multiple comparisons was performed and p-values are shown.

Article Snippet: Western Blot band quantification was carried out using Image Lab Software (Bio Rad).

Techniques: Fluorescence, Injection, Clear Native PAGE, Western Blot, ThT Assay, SDS Page, Cryo-EM Sample Prep, Immunolabeling, Immuno-Electron Microscopy, Labeling

Journal: bioRxiv

Article Title: Galectin-3 deletion modulates microglial phenotype and Aβ response via TREM2 activation while attenuating neuroinflammation

doi: 10.1101/2025.03.17.643790

Figure Lengend Snippet: A Representative Western Blot images of TLR4 and TREM2 after the different Aβ treatments. β-actin was used as housekeeping. B Quantification of TLR4 Western Blot bands (n≥4/group). C Quantification of TREM2 Western Blot bands (n≥4/group). D Representative individual plaque staining of microglial activation markers CD68 and TREM2, with ThS. E Proportion of plaque covered by CD68 or TREM2 in hippocampus (n=20 plaques per mouse, 5 mice/group). D Representative individual plaque staining of dystrophic neurite marker LAMP1 in APP and APP-Gal3KO mouse brains. E Quantification of individual plaque dystrophic neurite content in cortex and hippocampus (n=75 plaques per mouse, 5 mice/group). In B and C, values are expressed as individual experimental replicates with mean ±SEM. In E and G, values are expressed as violin plots. In B and C, two-way ANOVA with Tukey’s multiple comparisons was performed. In E and G, Mann-Whitney test was performed. P-values are expressed with 3 decimals.

Article Snippet: Western Blot band quantification was carried out using Image Lab Software (Bio Rad).

Techniques: Western Blot, Staining, Activation Assay, Marker, MANN-WHITNEY