primary monoclonal antibody against βapp 22c11  (Millipore)

Journal: Frontiers in Neuroscience

Article Title: Fool's gold standard? Immunoperoxidase staining with the mouse monoclonal antibody (Clone 22C11) for detecting axonal pathology after traumatic brain injury

doi: 10.3389/fnins.2025.1613172

Figure Lengend Snippet: Summary diagram illustrating targets in white matter tracts revealed by immunofluorescent vs. immunohistochemical staining with the N-Terminal antibody (22C11) and C-Terminal antibody (Y188) for amyloid precursor protein (APP). (A) Immunofluorescent staining is performed using fluorophore ( FP )-conjugated secondary antibodies (2°) that is visible under a microscope. An unknow antigen (Ag) in astrocytes can be specifically recognized by 22C11 , and APP in oligodendrocytes by Y188 . (B) Immunoperoxidase staining is performed using biotinylated secondary antibodies that needs further incubation with the ABC kit before an enzymatic reaction for the horseradish peroxidase ( HRP ) contained in the staining kit. Avidin (A) in the kit will bind to biotin (B) that is conjugated to the secondary antibodies, resulting in specific immunostaining. Avidin can also bind to endogenous biotin (B) that is present in astrocytes and oligodendrocytes. Therefore, both glial groups can be coincidently stained by the ABC kit. Astro, astrocyte; Olig, oligodendrocyte.

Article Snippet: While some researchers diluted 22C11 at 1:100–200 (Ryu et al., ; Xiong et al., , ) as recommended by the manufacturer (Millipore-Sigma), others used this primary antibody at a concentration as high as 1:80,000 or even 1:130,000 (Johnson et al., ; Koch et al., ).

Techniques: Immunohistochemical staining, Staining, Microscopy, Immunoperoxidase Staining, Incubation, Avidin-Biotin Assay, Immunostaining

Journal: iScience

Article Title: Human monocyte-derived macrophages shift subcellular metalloprotease activity depending on their activation state

doi: 10.1016/j.isci.2024.111171

Figure Lengend Snippet: Cellular entry may dictate intracellular MMP-9 localization in M(IL-4) (A) Representative immunofluorescent images of M(LPS/IFN-γ) and M(IL-4) over time (0 min–24 h) upon incubation with exogenous fluorescently labeled recombinant human MMP-9 (red). The structure of cells is visualized by F-actin staining (Phalloidin, green) and nuclei are visualized by Hoechst 33342 staining (DAPI, blue). Images are representative of 8 different donors. The scale bar represents 50 μm. (B) Quantification of MMP-9 uptake over time by M(LPS/IFN-γ) and M(IL-4) ( n = 8). Error bars are mean ± SEM. p values less than 0.05 were considered significant (∗∗ p < 0.01). (C) Quantification of immunofluorescent images of M(LPS/IFN-γ) and M(IL-4) after 6 h of incubation with fluorescently labeled MMP-9. The blue bar shows MMP-9 co-localization with the nucleus, and the green bar shows MMP-9 co-localization with F-actin (in the absence of nuclear co-localization) ( n = 8). Error bars are mean ± SEM. p values less than 0.05 were considered significant (∗∗ p < 0.01). (D) Representative confocal images of M(IL-4) incubated with fluorescently labeled recombinant human MMP-9. The nucleus is shown in blue (DAPI) and MMP-9 is shown in red. Regions of MMP-9/DAPI colocalization are shown in white. The top panel shows confocal images; the bottom panel shows a surface depiction of the confocal images. The scale bar represents 10 μm. (E) Confocal images of M(IL-4) incubated with exogenous fluorescently labeled recombinant human MMP-9. The nucleus is shown in blue (DAPI) and MMP-9 is shown in red. The top panel shows a surface depiction of MMP-9. The bottom panel shows a coronal cross section (XY plane) of the surface depiction of MMP-9. The scale bar represents 10 μm. (F) Representative immunofluorescent images of M(IL-4) incubated with exogenous fluorescently labeled recombinant MMP-9 (red, left panel), TIMP-1 (red, middle panel) and MMP-9/TIMP-1 complex (orange, right panel). The structure of cells is visualized by F-actin staining (Phalloidin, green) and nuclei are visualized by Hoechst 33342 staining (DAPI, blue). Images are representative of 7 different donors. The scale bar represents 50 μm. (G) Average uptake (between 30 min and 6 h) of exogenous fluorescently labeled recombinant MMP-9, TIMP-1, and MMP-9/TIMP-1 complex. Each data point represents cells from a different donor ( n = 7). Error bars are mean ± SEM. p values less than 0.05 were considered significant (∗ p < 0.05). (H) Flow cytometry analysis of LRP-1, CD63, CD74, and APP1 on M(M-CSF), M(LPS/IFN-γ) and M(IL-4). Each data point represents cells from a different donor (at least n = 9). Error bars are mean ± SEM. p -values less than 0.05 were considered significant (∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns non-significant). (I) Representative immunofluorescent images of M(IL-4) incubated with exogenous fluorescently labeled recombinant MMP-9 (red) in the presence of RAP (LRP-1 inhibitor). Structure of cells is visualized by F-actin staining (Phalloidin, green) and nuclei are visualized by Hoechst 33342 staining (DAPI, blue). Images are representative of 6 different donors. The scale bar represents 50 μm. (J) Average uptake (between 30 min and 6 h) of exogenous fluorescently labeled recombinant MMP-9 in the presence of RAP (LRP-1 inhibitor). Each data point represents cells from a different donor ( n = 6). Error bars are mean ± SEM. p values less than 0.05 were considered significant (ns non-significant). (K) Flow cytometry analysis of translocating protein Ku70/80 on M(M-CSF), M(LPS/IFN-γ), and M(IL-4). Each data point represents cells from a different donor ( n = 6). Error bars are mean ± SEM. p -values less than 0.05 were considered significant (∗ p < 0.05; ∗∗∗ p < 0.001). See also Figure S7 .

Article Snippet: Anti-APP1 Alexa Fluor 488 (clone 22C11) , Merck , Cat # MAB348A4; RRID: AB_11213747.

Techniques: Incubation, Labeling, Recombinant, Staining, Flow Cytometry

Journal: iScience

Article Title: Human monocyte-derived macrophages shift subcellular metalloprotease activity depending on their activation state

doi: 10.1016/j.isci.2024.111171

Figure Lengend Snippet:

Article Snippet: Anti-APP1 Alexa Fluor 488 (clone 22C11) , Merck , Cat # MAB348A4; RRID: AB_11213747.

Techniques: Plasmid Preparation, Recombinant, Knock-Out, Blocking Assay, Staining, Antibody Labeling, Reverse Transcription, Lysis, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Fractionation, Cell Culture, Software, Flow Cytometry, Fluorescence, Spectrophotometry