Nicotinic Acetylcholine Receptor epsilon Antibody

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• Nicotinic Acetylcholine Receptor epsilon Antibody

  Name: Andrea Salzinger

  Application: Immunocytochemistry

  Sample Tested: human NMJ containing organoid

  Species: Human

  Verified Customer | Posted 02/06/2022

  Immunofluorescent staining against epsilon subunit of Acetylcholine receptors. FastMyHc: Fast Myosin heavy chain (muscle fibres), Btx: Bungarotoxin (AChR alpha), AChRE: epsilon subunit of AChR.

  Human 3D organoids containing neuromuscular junctions (NMJs) were tested for the untested application IF staining of Acetylcholine receptor (AChR) subunit epsilon. NMJs were visualized with Bungarotoxin (Btx) which binds to the alpha subunit of the AChRs. Muscle fibres are shown with Fast Myosin Heavy Chain (FastMyHc). Antibody against AChR epsilon (NBP2-26108) did not co-localise with Btx staining on FastMyHc+ muscle fibres (Figure AChRE). Moreover, AChR epsilon Ab appears to bind to axons, as AChR epsilon staining is comparable to NfH axonal staining. Secondary only staining did not reveal any unspecific axonal binding of the secondary antibody, demonstrating that axonal like staining is owed to AChR epsilon Ab. Neuromuscular junction (NMJ) organoids were fixed in 4% PFA for 3h and subsequently washed 3x with PBS. Cryoprotection was carried out by incubation in 30% Sucrose overnight, followed by embedding in a 1:1 mixture of 30% Sucrose:OCT and cryosection into 20m slices, collected on superfrost glass slides. For IF staining, samples were incubated for 10min with 0.25% Triton X-100 to ensure membrane permeabilization, followed by blocking with 5% bovine serum albumin, 0.25% Triton X-100 in PBS for 1h, RT to avoid unspecific binding of primary antibodies. Subsequently, samples were incubated with primary antibodies in blocking solution overnight, 4°C. The following day, samples were washed 2x with PBS and 1x with PBS-T (0.1% Tween 20) and incubated with appropriate fluorescent secondary antibodies for 1h, RT. Nuclei were counter stained with DAPI for 10min, followed by subsequent washing with 2x PBS and 1x PBS-T. Samples were mounted using Fluorosave. Images were taken with confocal microscope and analysed.

  
  Bio-Techne Response

  This review was submitted through the legacy Novus Innovators Program, reflecting a new species or application tested on a primary antibody.

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