Hpa014784

Manufactured by Merck Group

HPA014784 is a laboratory equipment product from Merck Group. It is designed for general scientific applications, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Additional information about the intended use or features of this product is not available.

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Lab products found in correlation

Autostainer link 48, by Agilent Technologies (2 mentions) Mca1477, by Bio-Rad (2 mentions) K5001, by Agilent Technologies (2 mentions) Clone m3 84, by BD (2 mentions) Lsm800 confocal microscope, by Zeiss (1 mentions) Matrigel, by Corning (1 mentions) G3893, by Merck Group (1 mentions) M7020, by Agilent Technologies (1 mentions) Ab19857, by Abcam (1 mentions) Hpa015768, by Merck Group (1 mentions) Vectashield containing dapi, by Vector Laboratories (1 mentions)

3 protocols using hpa014784

Quantifying Perivascular AQP4 Loss

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Mice were sacrificed under deep anesthesia by intracardial perfusion with PBS followed by perfusion with 4% w/v paraformaldehyde solved in PBS. Brains, spinal cords, optic nerves, kidneys and spleens were removed and fixed in 4% paraformaldehyde overnight. Cervical, thoracic, and lumbar spinal cord was cut into 11–12 4 mm thick transverse segments prior to embedding; 5 μm thick sections were stained for hematoxylin and eosin (H&E) and Luxol-fast blue/periodic acid-Schiff. Immunohistochemistry was performed using a biotin-streptavidin peroxidase technique (K5001; Dako) and an automated immunostainer (AutostainerLink 48; Dako). Sections were pretreated in a steamer (treatment solutions pH 6.0 or 9.0; Dako) before incubation with the primary antibodies anti-Mac3 (clone M3/84, 553322, 1:100; BD Pharmingen), anti-CD3 (MCA 1477, 1:50; Serotec), and anti-AQP4 (HPA014784, 1: 4000; Sigma). DAB was used as a chromogen and sections were counterstained using hematoxylin.
To quantify the perivascular loss of immunoreactivity against AQP4, three consecutive brain sections per mouse were analyzed using Image J (NIH) [49 (link)]. The sizes of areas showing AQP4 loss and the corresponding vessel lumen were measured and processed as individual ratios (any ratio > 1 indicates perivascular AQP4 loss).

Vogel A., Knier B., Lammens K., Kalluri S.R., Kuhlmann T., Bennett J.L, & Korn T. (2017). Deletional tolerance prevents AQP4‐directed autoimmunity in mice. European Journal of Immunology, 47(3), 458-469.

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Quantifying Perivascular AQP4 Loss

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Mice were sacrificed under deep anesthesia by intracardial perfusion with PBS followed by perfusion with 4% w/v paraformaldehyde solved in PBS. Brains, spinal cords, optic nerves, kidneys and spleens were removed and fixed in 4% paraformaldehyde overnight. Cervical, thoracic, and lumbar spinal cord was cut into 11–12 4 mm thick transverse segments prior to embedding; 5 μm thick sections were stained for hematoxylin and eosin (H&E) and Luxol‐fast blue/periodic acid‐Schiff. Immunohistochemistry was performed using a biotin‐streptavidin peroxidase technique (K5001; Dako) and an automated immunostainer (AutostainerLink 48; Dako). Sections were pretreated in a steamer (treatment solutions pH 6.0 or 9.0; Dako) before incubation with the primary antibodies anti‐Mac3 (clone M3/84, 553322, 1:100; BD Pharmingen), anti‐CD3 (MCA 1477, 1:50; Serotec), and anti‐AQP4 (HPA014784, 1: 4000; Sigma). DAB was used as a chromogen and sections were counterstained using hematoxylin.
To quantify the perivascular loss of immunoreactivity against AQP4, three consecutive brain sections per mouse were analyzed using Image J (NIH) 49. The sizes of areas showing AQP4 loss and the corresponding vessel lumen were measured and processed as individual ratios (any ratio > 1 indicates perivascular AQP4 loss).

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Comprehensive Immunofluorescence Characterization of iPSCs, NPCs, and Astrocytes

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iPSC, NPC and astrocyte cultures were first plated in Matrigel (Corning, USA)‐coated Lab‐tek 8‐well chamber slide systems (Thermo Fisher, USA) at a density of 4 × 10⁴ cells per well, then fixed with 4% PFA, followed by permeabilization with 0.01% Triton X‐100, and blocking with 5% BSA in PBS. The cells were then incubated overnight with the following primary or conjugated antibodies: rabbit anti‐OCT4 (ab19857, Abcam, 1:100); APC anti‐SSEA4 (FAB1435A, R&D Systems, 1:100); AF647 anti‐nestin (560,393, BD, 1:100); mouse anti‐GFAP (G3893, Sigma, 1:100); mouse anti‐Vimentin (M7020, DAKO, 1:100); rabbit anti‐S100‐β (HPA015768, Sigma, 1:100); rabbit anti‐AQP4 (HPA014784, Sigma, 1:100) at 4°C, and subsequently incubated with the following secondary antibodies: AF488 goat anti‐rabbit IgG (A11034, Life, 1:250); AF488 goat anti‐mouse IgG (A11001, Life, 1:250); AF594 donkey anti‐mouse IgG (A21203, Life, 1:250); AF594 donkey anti‐rabbit IgG (A21207 Life, 1:250) for 1h at room temperature. Slides were mounted with Vectashield containing DAPI (Vector Laboratories, USA), dried, sealed, and the analysis was performed using a Zeiss LSM 800 confocal microscope. Quantification of the percentage of positive cells was performed using ImageJ software.

Ghirotto B., Oliveira D.F., Cipelli M., Basso P.J., de Lima J., Breda C.N., Ribeiro H.C., Silva C.C., Sertié A.L., Oliveira A.E., Hiyane M.I., Caldini E.G., Sussulini A., Nakaya H.I., Kowaltowski A.J., Oliveira E.M., Zatz M, & Câmara N.O. (2022). MS‐Driven Metabolic Alterations Are Recapitulated in iPSC‐Derived Astrocytes. Annals of Neurology, 91(5), 652-669.

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