polyclonal goat anti mouse il 33  (R&D Systems)

Journal: Molecular Psychiatry

Article Title: Requirement of brain interleukin33 for aquaporin4 expression in astrocytes and glymphatic drainage of abnormal tau

doi: 10.1038/s41380-020-00992-0

Figure Lengend Snippet: a , b Micrographs are reverted monochrome for FITC channel of AQP4 staining in the cerebral cortex a or hippocampus b in WT or Il33 −/− mice, revealing significant reduction of AQP4 after 60 weeks in Il33 −/− brains. CA , cornu ammonis; DG , dentate gyrus. c Statistical summary for immunofluorescence-based detection of AQP4 expression level demonstrates a significant reduction of AQP4 in both cerebral cortex and hippocampus at/after 60 weeks as compared with WT mice. IOD , integrated optical density. d Immunofluorescence shows disappearance of n-AQP4 in Il33 −/− brain at or after 60 weeks; note the presence of tubular/linear p-APQ4 (arrows) in Il33 −/− brains at 60 and 75 weeks; numerous age-related pigments in 75-week Il33 −/− brain are indicated by arrowheads. Silver staining (lower panels) demonstrates substantial numbers of neurites in Il33 −/− brain at 60 weeks, and nearly none at 75 weeks. e RT-PCR on cortical RNA shows a reduction of Aqp4 mRNA in Il33 −/− brains after 45 weeks. f Western blot of the cortical proteins shows reduction of AQP4 (green) in Il33 −/− mice at 65 weeks; α-actin (red) was used as internal control and probed simultaneously; its staining saturation is revealed (white) for more accurate evaluation. Bars’ unit = μm.

Article Snippet: Following antibodies were used in this study: biotin goat anti-mouse IL33 (R&D System, Minneapolis, MN, USA), monoclonal rat anti-mouse IL33 (clone 396118, ProSci, Charleston, SC, USA), rabbit anti-glial fibrillary acidic protein (GFAP) (astrocyte marker, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-tubulin β3 (neurons, R&D system), mouse monoclonal antibody AT8, which recognizes phosphor-tau(Ser202/Thr205) (ThermoFisher, Waltham, MA, USA), mouse monoclonal antibodies to PHF1 tau (Sigma-Aldrich), FITC-labeled or non-labeled anti-α-actin (Sigma-Aldrich), and rabbit anti-AQP4 antibody (Millipore).

Techniques: Staining, Immunofluorescence, Expressing, Silver Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Molecular Psychiatry

Article Title: Requirement of brain interleukin33 for aquaporin4 expression in astrocytes and glymphatic drainage of abnormal tau

doi: 10.1038/s41380-020-00992-0

Figure Lengend Snippet: a Immunofluorescence shows more PHF1 (green) in glomeruli of WT mice than Il33 −/− littermates at 70 weeks; right panels are enlarged boxed areas of left panels to show PHF1 in cytoplasm of glomerular mesangial cells (arrows). b Electron microscopy shows structural relationship of mesangial cells (MC) with other cells and glomerular basement membrane (GBM). c Western blot shows PHF1 in the cerebral cortex and isolated glomeruli of WT or Il33 −/− mice at 70 weeks; note an additional band of low molecular weight of PHF1 (arrow) among glomerular proteins, which is absent in cortical proteins. d RT-PCR shows a lack of tau mRNA in glomeruli as compared to the cortex in both WT and Il33 −/− mice. e Immunohistochemistry on PHF1 in the cerebral cortical and glomerular tissue from the same representative WT or Il33 −/− mice reveals a reverse correlation between PHF1 in the two tissue locations, i.e., more neuronal PHF1 vs less glomerular PHF1 (arrows) in Il33 −/− mice, and vice versa in WT mice. f Statistical analysis shows a positive regression between glomerular PHF1 and cortical AQP4 among WT and Il33 −/− mice. IOD , integrated optical density. g Immunofluorescence on normal human kidneys reveals the presence of PHF1, but not phospho-tau(Ser202/Thr205) (AT8), in glomeruli; an isotype IgG control is also shown; numbered boxed areas are enlarged to show PHF1 in mesangial cells. Bars’ unit = μm.

Article Snippet: Following antibodies were used in this study: biotin goat anti-mouse IL33 (R&D System, Minneapolis, MN, USA), monoclonal rat anti-mouse IL33 (clone 396118, ProSci, Charleston, SC, USA), rabbit anti-glial fibrillary acidic protein (GFAP) (astrocyte marker, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-tubulin β3 (neurons, R&D system), mouse monoclonal antibody AT8, which recognizes phosphor-tau(Ser202/Thr205) (ThermoFisher, Waltham, MA, USA), mouse monoclonal antibodies to PHF1 tau (Sigma-Aldrich), FITC-labeled or non-labeled anti-α-actin (Sigma-Aldrich), and rabbit anti-AQP4 antibody (Millipore).

Techniques: Immunofluorescence, Electron Microscopy, Membrane, Western Blot, Isolation, Molecular Weight, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Control

Journal: Molecular Psychiatry

Article Title: Requirement of brain interleukin33 for aquaporin4 expression in astrocytes and glymphatic drainage of abnormal tau

doi: 10.1038/s41380-020-00992-0

Figure Lengend Snippet: a Immunofluorescence shows a significant increase in p-AQP4 (red) expression in the cortex of Il33 −/− mice after receiving rIL33 injection ( Il33 −/− +rIL33) as compared with PBS controls ( Il33 −/− +PBS); two-color staining (lower panels) reveals high density of p-AQP4 (red) but nearly no granular n-APQ4 around neurons (green, tubulinβ3) in Il33 −/− +rIL33 mice, in contrast to many (arrows) in WT mice. Also note much less tubulinβ3 in both Il33 −/− +rIL33 and Il33 −/− +PBS mice than WT mice. b Immunofluorescence shows an increase in p-AQP4 expression in hippocampus (upper panels) of Il33 −/− +rIL33 mice; note that GFAP + astrocyte (green) population is similar among three groups (lower panel). c Western blot on cortical proteins shows quantity of AQP4 in Il33 −/− +rIL33 mice is comparable to WT mice, and much higher than Il33 −/− +PBS controls. d , e Statistics on total AQP4 expression (% of area) ( d ), and numbers of AQP4 + regions ( e ) in the cortex demonstrates that rIL33 injection enhances total AQP4 expression level but not numbers of AQP4 + regions.

Article Snippet: Following antibodies were used in this study: biotin goat anti-mouse IL33 (R&D System, Minneapolis, MN, USA), monoclonal rat anti-mouse IL33 (clone 396118, ProSci, Charleston, SC, USA), rabbit anti-glial fibrillary acidic protein (GFAP) (astrocyte marker, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-tubulin β3 (neurons, R&D system), mouse monoclonal antibody AT8, which recognizes phosphor-tau(Ser202/Thr205) (ThermoFisher, Waltham, MA, USA), mouse monoclonal antibodies to PHF1 tau (Sigma-Aldrich), FITC-labeled or non-labeled anti-α-actin (Sigma-Aldrich), and rabbit anti-AQP4 antibody (Millipore).

Techniques: Immunofluorescence, Expressing, Injection, Staining, Western Blot

Journal: Molecular Psychiatry

Article Title: Requirement of brain interleukin33 for aquaporin4 expression in astrocytes and glymphatic drainage of abnormal tau

doi: 10.1038/s41380-020-00992-0

Figure Lengend Snippet: a Immunohistochemistry shows neuronal PHF1 in indicated groups; note slightly reduced PHF1 in Il33 −/− +rIL33 mice as compared with Il33 −/− +PBS controls; no PHF1 was detectable in WT mice. Bars’ unit = μm b Regression analysis shows no linear correlation between total cortical AQP4 level (% of area) vs neuronal PHF1 among Il33 −/− +PBS, Il33 −/− +IL33, and WT mice; note that total AQP4 level in Il33 −/− +IL33 mice is not proportional to neuronal PHF1. Neuronal PHF1 was quantitated on digital immunostaining imagines, and expressed as IOD/neurons. c Statistical analysis shows a linear negative regression (black dotted line) between cortical and glomerular PHF1 among WT , Il33 −/− +PBS and Il33 −/− +rIL33 mice at 70 weeks; three mice were used for each group. d ELISA shows a sixfold increase in serum pCol peptide that had been injected into ventricles 24 h ago in Il33 −/− +rIL33 mice, as compared to PBS controls. e Diagram depicts two aqueous flows driven by astrocyte AQP4 (lightly colored arrows): Flow 1, driven by n-AQP4, brings neuronal wastes away from neurons, and Flow 2, convective flow driven by p-AQP4 toward perivenous space, flushes brain wastes further to perivenous space, i.e., glymphatics. Therefore, a lack of n-AQP4 results in ineffective removal of neuronal wastes, e.g., PHF1 from neurons.

Article Snippet: Following antibodies were used in this study: biotin goat anti-mouse IL33 (R&D System, Minneapolis, MN, USA), monoclonal rat anti-mouse IL33 (clone 396118, ProSci, Charleston, SC, USA), rabbit anti-glial fibrillary acidic protein (GFAP) (astrocyte marker, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-tubulin β3 (neurons, R&D system), mouse monoclonal antibody AT8, which recognizes phosphor-tau(Ser202/Thr205) (ThermoFisher, Waltham, MA, USA), mouse monoclonal antibodies to PHF1 tau (Sigma-Aldrich), FITC-labeled or non-labeled anti-α-actin (Sigma-Aldrich), and rabbit anti-AQP4 antibody (Millipore).

Techniques: Immunohistochemistry, Immunostaining, Enzyme-linked Immunosorbent Assay, Injection