Immunohistochemical Analysis of Muscle, Spinal Cord, and Optic Nerve

Cryosections of tibialis anterior muscles were incubated with rabbit anti–mouse AQP4 (1:200; catalog A5971, Sigma-Aldrich) and mouse anti-DDK (FLAG, 1:500; catalog TA50011-100, OriGene) at 4°C overnight. Cryosections of spinal cords and optic nerves were incubated with the following primary antibodies at 4°C overnight: rabbit anti–zonula occludens 1 (anti–ZO-1; 1:200; catalog AB216880, Abcam), goat anti–mouse IgG (1:100; catalog AB6708, Abcam), rabbit anti-AQP4 (1:200; catalog A5971, Sigma-Aldrich), mouse anti–glial fibrillary acidic protein (anti-GFAP; 1:200; catalog SC33673, Santa Cruz Biotechnology), rabbit anti–oligodendrocyte transcription factor 2 (anti-Olig2; 1:200; catalog AB9610, Millipore), goat anti–myelin basic protein (anti-MBP; 1:200; catalog SC13914, Santa Cruz Biotechnology), rabbit anti–neurofilament-heavy (anti–NF-H; 1:400; catalog N4142, Sigma-Aldrich), rabbit anti–ionized calcium–binding adapter molecule 1 (anti–Iba-1; 1:200; catalog 019-19741, Wako), rabbit anti–terminal complement complex (anti–C5b-9; 1:200; catalog AB55811, Abcam), rat anti–lymphocyte antigen 6 complex locus G6D (anti-Ly6G; 1:400; catalog AB2557, Abcam), rat anti–Siglec-F (1:50; catalog 552125, BD Biosciences), mouse anti–cluster of differentiation 68 (anti-CD68; 1:200; catalog YM3161, Immunoway), and rat anti-F4/80 (1:200; catalog AB6640, Abcam). Sections were then incubated with the appropriate Alexa Fluor–conjugated secondary antibodies (Thermo Fisher Scientific) at room temperature for 1 hour. Neuronal cells were visualized using blue fluorescent Nissl stain (NeuroTrace, Thermo Fisher Scientific). Sections were coverslipped with antifade reagent containing DAPI (Thermo Fisher Scientific). Selected sections were stained with H&E and Luxol fast blue using standard procedures.

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Yick L.W., Ma O.K., Chan E.Y., Yau K.X., Kwan J.S, & Chan K.H. (2023). T follicular helper cells contribute to pathophysiology in a model of neuromyelitis optica spectrum disorders. JCI Insight, 8(4), e161003.

Publication 2023

Anti igg Antibodies C5b 9 Calcium Cells Cryosections Dapi Gfap Goat Luxol fast blue Lymphocyte antigen Mouse Myelin basic protein Neurofilament Neuronal Olig2 Optic nerves Rabbit Siglec 1 Spinal cords Tibialis anterior muscles Zonula occludens

Corresponding Organization :

Other organizations : University of Hong Kong, Chinese University of Hong Kong

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Variable analysis

independent variables

Incubation with primary antibodies: rabbit anti–mouse AQP4 (1:200), mouse anti-DDK (FLAG, 1:500), rabbit anti–zonula occludens 1 (anti–ZO-1; 1:200), goat anti–mouse IgG (1:100), rabbit anti-AQP4 (1:200), mouse anti–glial fibrillary acidic protein (anti-GFAP; 1:200), rabbit anti–oligodendrocyte transcription factor 2 (anti-Olig2; 1:200), goat anti–myelin basic protein (anti-MBP; 1:200), rabbit anti–neurofilament-heavy (anti–NF-H; 1:400), rabbit anti–ionized calcium–binding adapter molecule 1 (anti–Iba-1; 1:200), rabbit anti–terminal complement complex (anti–C5b-9; 1:200), rat anti–lymphocyte antigen 6 complex locus G6D (anti-Ly6G; 1:400), rat anti–Siglec-F (1:50), mouse anti–cluster of differentiation 68 (anti-CD68; 1:200), and rat anti-F4/80 (1:200)

dependent variables

Localization and expression of target proteins (AQP4, DDK, ZO-1, GFAP, Olig2, MBP, NF-H, Iba-1, C5b-9, Ly6G, Siglec-F, CD68, F4/80) in cryosections of tibialis anterior muscles, spinal cords, and optic nerves

control variables

Cryosections of tibialis anterior muscles, spinal cords, and optic nerves
Incubation of primary antibodies at 4°C overnight
Incubation of Alexa Fluor–conjugated secondary antibodies at room temperature for 1 hour
Use of Nissl stain for visualization of neuronal cells
Coverslipping of sections with antifade reagent containing DAPI
Staining of selected sections with H&E and Luxol fast blue

positive controls

Not specified

negative controls

Not specified

Annotations

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