Middle frontal neocortex was dounce homogenized using pestil B (Kimble Chase, Rockwood, TN, USA) in 0.25M sucrose in TKM (50mM Tris, 25mM KCl, 5 mM MgCl2) buffer. The homogenate was adjusted to 1.6M using 2.3M sucrose in TKM. The homogenate was spun on a 1.8M sucrose cushion in TKM using a SW41 rotor on the Beckman Coulter XPN-80 ultracentrifuge at 40,000 g for 40 minutes at 4 C (Beckman Coulter Inc, Indianapolis, IN, USA). Isolated nuclei were stained with Alexa Fluor 647 conjugated to 2089 (rabbit polyclonal C-terminal anti-TDP-43 antibody, Center for Neurodegenerative Disease Research, University of Pennsylva-nia), Alexa Fluor 488 conjugated to NeuN (EMD Millipore, Billerica, MA, USA), and DAPI (Invitrogen, Carlsbad, CA, USA). Alexa Fluor 647 was conjugated to 2089 according to the APEX Alexa Fluor 647 Antibody labeling kit protocol (Thermo Fisher Scientific, Waltham, MA, USA). Stained nuclei were sorted for single cells based on DAPI, NeuN and TDP-43 fluorescence on the BD FACSAria II (BD Biosciences, San Jose, CA, USA) at 20 psi on 100mm nozzle.
Pestil b
Manufactured by DWK Life Sciences
Sourced in United States
The Pestil B is a laboratory mortar and pestle set designed for grinding and mixing solid materials. It consists of a bowl (Pestil) and a hand-operated grinding tool (Pistil). The core function of the Pestil B is to facilitate the mechanical reduction of sample sizes and the homogenization of solid substances for further analysis or processing in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Xpn 80, by Beckman Coulter (4 mentions)
Apex alexa fluor 647 antibody labeling kit protocol, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Facsaria 2, by BD (4 mentions)
Alexa fluor 488 conjugated to neun, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (2 mentions)
Alexa fluor 488 conjugated neun, by Merck Group (2 mentions)
4 protocols using pestil b
1
Nuclei Isolation and TDP-43 Profiling
2
TDP-43 and NeuN Positive Nucleus Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Human autopsy tissue was obtained from the University of Pennsylvania Center for Neurodegenerative Disease Research Brain Bank as described (53 (link)). Informed consent from next of kin was obtained for every case.
Isolation was performed exactly as previously described (52 (link)). Briefly, mid-frontal neocortex of all cases were dounce homogenized using pestil B (Kimble Chase) in 0.25M sucrose and adjusted to final molarity of 1.6M sucrose in TKM. The homogenate was spun on a 1.8M sucrose cushion on the Beckman Coulter XPN-80 ultracentrifuge at 40,000g for 40 minutes at 4°C (Beckman Coulter Inc, Indianapolis, IN, USA). Isolated nuclei were stained with Alexa Fluor 647 conjugated to 2089 (rabbit polyclonal C-terminal anti-TDP-43 antibody, Center for Neurodegenerative Disease Research, University of Pennsylvania), Alexa Fluor 488 conjugated NeuN (EMD Millipore, Billerica, MA, USA), and DAPI (Invitrogen, Carlsbad, CA, USA). Alexa Fluor 647 was conjugated to 2089 according to the APEX Alexa Fluor 647 Antibody labeling kit protocol (Thermo Fisher Scientific, Waltham, MA, USA). Stained nuclei were sorted for single cells containing TDP-43 and NeuN on the BD FACSAria II (BD Biosciences, San Jose, CA, USA) at 20 psi on 100μm nozzle.
Isolation was performed exactly as previously described (52 (link)). Briefly, mid-frontal neocortex of all cases were dounce homogenized using pestil B (Kimble Chase) in 0.25M sucrose and adjusted to final molarity of 1.6M sucrose in TKM. The homogenate was spun on a 1.8M sucrose cushion on the Beckman Coulter XPN-80 ultracentrifuge at 40,000g for 40 minutes at 4°C (Beckman Coulter Inc, Indianapolis, IN, USA). Isolated nuclei were stained with Alexa Fluor 647 conjugated to 2089 (rabbit polyclonal C-terminal anti-TDP-43 antibody, Center for Neurodegenerative Disease Research, University of Pennsylvania), Alexa Fluor 488 conjugated NeuN (EMD Millipore, Billerica, MA, USA), and DAPI (Invitrogen, Carlsbad, CA, USA). Alexa Fluor 647 was conjugated to 2089 according to the APEX Alexa Fluor 647 Antibody labeling kit protocol (Thermo Fisher Scientific, Waltham, MA, USA). Stained nuclei were sorted for single cells containing TDP-43 and NeuN on the BD FACSAria II (BD Biosciences, San Jose, CA, USA) at 20 psi on 100μm nozzle.
3
TDP-43 and NeuN Positive Nucleus Isolation
4
Nuclei Isolation and TDP-43 Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Middle frontal neocortex was dounce homogenized using pestil B (Kimble Chase, Rockwood, TN, USA) in 0.25M sucrose in TKM (50mM Tris, 25mM KCl, 5 mM MgCl2) buffer. The homogenate was adjusted to 1.6M using 2.3M sucrose in TKM. The homogenate was spun on a 1.8M sucrose cushion in TKM using a SW41 rotor on the Beckman Coulter XPN-80 ultracentrifuge at 40,000 g for 40 minutes at 4 C (Beckman Coulter Inc, Indianapolis, IN, USA). Isolated nuclei were stained with Alexa Fluor 647 conjugated to 2089 (rabbit polyclonal C-terminal anti-TDP-43 antibody, Center for Neurodegenerative Disease Research, University of Pennsylva-nia), Alexa Fluor 488 conjugated to NeuN (EMD Millipore, Billerica, MA, USA), and DAPI (Invitrogen, Carlsbad, CA, USA). Alexa Fluor 647 was conjugated to 2089 according to the APEX Alexa Fluor 647 Antibody labeling kit protocol (Thermo Fisher Scientific, Waltham, MA, USA). Stained nuclei were sorted for single cells based on DAPI, NeuN and TDP-43 fluorescence on the BD FACSAria II (BD Biosciences, San Jose, CA, USA) at 20 psi on 100mm nozzle.
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