HB9, a homeobox gene, is expressed in embryonic MNs early after differentiation from neuronal progenitors.18 , 19 An Hb9‐eGFP ES cell line on a pure C57Bl6 background, denoted as HBGB6, was generously provided by Dr. Craig Cox (Jackson, ME) and was used to generate MNs. ESCMNs were generated by treating free‐floating clusters of ES cells known as embryonic bodies (EB) with retinoic acid (1 μmol/L; Sigma Aldrich, Oakville, ON, Canada) and smoothened agonist (500 nmol/L; Enzo Life Sciences, Farmingdale, NY, USA) as described previously.7 , 13 , 14 Differentiated EBs were enzymatically dissociated in Tryple express (ThermoFisher Scientific, Ottawa, ON, Canada) with 0.01% (w/v) DNaseI (Sigma‐Aldrich). The obtained single cell suspension was resuspended in DFK10.14 For in vitro experiments, 105 dissociated cells were plated onto growth factor reduced matrigel (BD Biosciences, Rockville, MD, USA)‐coated glass coverslips (ThermoFisher Scientific). In vitro cells were maintained in DFK10 supplemented with 10 ng/mL GDNF (Millipore, Etobicoke, ON, Canada) and 10 ng/mL CNTF (ThermoFisher Scientific). Media was changed every other day.
Coated glass coverslips
Manufactured by Thermo Fisher Scientific
Sourced in United States
Coated glass coverslips are flat, thin glass sheets used to cover and protect samples in microscopy applications. The glass surface is coated with a thin layer of a specific material to enhance the sample attachment or visualization. The core function of coated glass coverslips is to provide a clean, uniform, and compatible surface for mounting and observing samples under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Smoothened agonist, by Enzo Life Sciences (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Retinoic acid, by Merck Group (1 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Mouse serum, by Thermo Fisher Scientific (1 mentions)
Blocking reagent, by Thermo Fisher Scientific (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
2 protocols using coated glass coverslips
1
Generation of Hb9-eGFP Motor Neurons
2
Comprehensive Immunofluorescence Imaging of Murine Spleen
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2-3mm thick spleen sections were fixed with fresh 4% paraformaldehyde at 4°C for 4 h, followed by incubating in increasing sucrose concentrations from 10%-40% at 4°C. After overnight incubation in 40% sucrose, tissue was rapidly frozen in embedding media (Tissue-Tek® O.C.T; Sakura), left overnight at -80°C, and then stored at -20°C. Unfixed tissue was immediately frozen in embedding media, left overnight at -80°C, and then stored at -20°C. 10-14 µm thick cryosections were cut, put on coated glass cover slips (Thermo Scientific) and dried in a desiccator. After blocking with 1% blocking reagent (Invitrogen) and 2% mouse serum (Invitrogen) in PBST (0.05% Tween in 1X PBS to amplify the signal. Sections were mounted in fluorescence mounting medium (Dako or Vectashield), and examined with a Zeiss LSM 700 confocal microscope using glycerol 25X, 40X, and 63X objectives. Tiles covering an entire spleen section were stitched together. High resolution images were obtained with a Zeiss LSM 800 using Airy Scanning.
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