Coronal sections containing primary motor cortex [30 ] were collected from each animal. Free floating sections were first washed twice for ten minutes in 0.01 M PBS before a blocking solution containing 1% normal goat serum and 0.3% triton-X (Sigma Aldrich USA) in 0.01 M PBS was added for one hour. Sections were incubated with anti-GFP antibody (1:2000, Nacalai Tesque, Cat# 04404-84, Kyoto, Japan), anti-hTDP-43 antibody (1:1000 Proteintech Cat# 60019-2-Ig, Rosemont, IL, USA) diluted in 0.3% triton-X in 0.01 M PBS. Sections were washed three times for ten minutes in PBS before being incubated with anti-rat AlexaFluor 488 secondary antibody, anti-mouse AlexaFluor 568 secondary antibody and DAPI (Invitrogen, Boston, MA, USA) for 90 min. All incubations were performed at 21 °C. Sections were mounted onto slides and coverslipped with PermaFluor (Boston, MA, USA) aqueous mounting media.
Anti gfp antibody
Manufactured by Nacalai Tesque
Sourced in Japan
The Anti-GFP antibody is a laboratory reagent used for the detection and analysis of green fluorescent protein (GFP) in various applications. It is a highly specific antibody that binds to the GFP molecule, allowing researchers to identify and quantify the presence of GFP in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti ctip2, by Abcam (2 mentions)
Anti cleaved caspase 3, by BD (2 mentions)
Triton x, by Merck Group (1 mentions)
Anti rat alexafluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexafluor 568 secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti histone h3 antibody, by Cell Signaling Technology (1 mentions)
Anti gst antibody, by Nacalai Tesque (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Protein a sepharose, by GE Healthcare (1 mentions)
Anti phospho histone h3, by Merck Group (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Anti tbr2, by Abcam (1 mentions)
Anti ki 67, by Leica camera (1 mentions)
Anti pax6, by Fortrea (1 mentions)
Anti ki67, by Thermo Fisher Scientific (1 mentions)
Anti hop, by Santa Cruz Biotechnology (1 mentions)
Anti tbr2, by R&D Systems (1 mentions)
Anti brn2, by Santa Cruz Biotechnology (1 mentions)
6 protocols using anti gfp antibody
1
Immunohistochemical Analysis of Motor Cortex
2
Immunoblotting Analysis of Protein Levels
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Immunoblotting was performed according to the standard protocol [22 (link)]. Anti-p65 antibody, anti-GFP antibody (04363–24; Nacalai Tesque, Kyoto, Japan), anti-GAPDH (#2118; Cell Signaling Technology), anti-Histone H3 antibody (#4499; Cell Signaling Technology) and anti-GST antibody (04435–84; Nacalai Tesque) were used as primary antibodies, and alkaline phosphatase-conjugated goat anti-mouse and rabbit IgG antibody (Sigma) were used as secondary antibodies. The levels of protein expression were determined by densitometry analysis of immunoblots using Image J software (1.49v; U.S. National Institutes of Health).
3
Overexpression and Detection of RsTRICΔC8 Mutants
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The GFP-tagged RsTRICΔC8 and its mutants were overeproduced in E. coli Rosetta2 (DE3) cells. DSS was added to the solubilized and membrane samples. After incubation at room temperature for 30 min, the products were fractionated by SDS-PAGE and detected by western blot analysis, using an anti-GFP antibody (Nacalai Tesque). Details are provided in the Supplementary information, Data S1 .
4
Affinity Purification of GFP- and FLAG-tagged Proteins
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The TBC1D9-GFP expression vector, RUTBC2-GFP expression vector, 3×FLAG-Rab7, 3×FLAG-Rab9
and 3×FLAG-Rab9 (Q67L) expression vectors were transfected in 293T cells. At 1 d
post-transfection, the cells were lysed by cell lysis buffer as previously described
[23 (link)]. Three hundred µg of
cellular protein was incubated with anti-FLAG antibody (Sigma-Aldrich) for 1 h at 4°C, and
precipitated for 1 h at 4°C with pre-cleared Protein A Sepharose (GE-Healthcare). Pellets
were washed in Tris-buffered saline and used for SDS-PAGE. Immunoblotting was performed
using anti-GFP antibody (Nacalai Tesque). Twenty µg of cellular protein
was also immunoblotted using antibodies against GFP and FLAG.
and 3×FLAG-Rab9 (Q67L) expression vectors were transfected in 293T cells. At 1 d
post-transfection, the cells were lysed by cell lysis buffer as previously described
[23 (link)]. Three hundred µg of
cellular protein was incubated with anti-FLAG antibody (Sigma-Aldrich) for 1 h at 4°C, and
precipitated for 1 h at 4°C with pre-cleared Protein A Sepharose (GE-Healthcare). Pellets
were washed in Tris-buffered saline and used for SDS-PAGE. Immunoblotting was performed
using anti-GFP antibody (Nacalai Tesque). Twenty µg of cellular protein
was also immunoblotted using antibodies against GFP and FLAG.
5
Immunohistochemistry of Neural Development
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Immunohistochemistry was performed as described previously with slight modifications (Toda et al., 2013 (link); Kawasaki et al., 2000 (link)). Coronal sections were permeabilized with 0.3% Triton X-100/PBS and incubated overnight with primary antibodies, which included anti-Tbr2 (Abcam, UK, RRID: AB_778267 ), anti-Pax6 (Covance, Princeton, NJ, RRID: AB_291612 ), anti-Ki-67 (Leica, Germany, RRID: AB_442102 ), anti-phospho-histone H3 (Millipore, Billerica, MA, RRID: AB_310016 ), anti-phosphorylated vimentin (Medical and Biological Laboratories, Japan, RRID: AB_592963 ), anti-cleaved caspase 3 (BD Pharmingen, San Diego, CA, RRID: AB_397274 ), anti-Ctip2 (Abcam, UK, RRID: AB_2064130 ), anti-FOXP2 (Atlas antibodies, Sweden, RRID: AB_1078908 ), anti-GFAP (Sigma-Aldrich, St. Louis, MO, RRID: AB_477010 ) and anti-GFP antibodies (Nacalai tesque, Japan, RRID: AB_2313652 ; Medical and Biological Laboratories, Japan, RRID: AB_591819 ). After incubation with secondary antibodies and Hoechst 33342, the sections were washed and mounted.
For triple immunostaining, after double immunostaining was performed as described above, the sections were incubated with biotin-conjugated anti-phospho-histone H3 antibody (Millipore, Billerica, MA, RRID:AB_310794 ) and subsequently with fluorescent-dye conjugated streptavidin.
For triple immunostaining, after double immunostaining was performed as described above, the sections were incubated with biotin-conjugated anti-phospho-histone H3 antibody (Millipore, Billerica, MA, RRID:
6
Immunohistochemistry of Neural Progenitor Markers
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Immunohistochemistry was performed as described previously with slight modifications (Kawasaki et al., 2000 (link); Toda et al., 2013 (link)). Sections were permeabilized with 0.3% Triton X-100 in PBS and blocked with 2% bovine serum albumin (BSA), 0.3% Triton X-100 in PBS. For quadruple-staining and immunostaining with Ki-67, sections were subjected to microwave antigen retrieval in a sodium citrate solution. The sections were then incubated overnight with primary antibodies, which included anti-Hop (Santa Cruz Biotechnology, RRID:AB_2687966 ), anti-HopX (Atlas Antibodies, RRID:AB_10603770 ), anti-Tbr2 (R&D Systems, RRID:AB_10569705 ; Abcam, RRID:AB_778267 ), anti-Pax6 (Millipore, RRID:AB_1587367 ), anti-Ki-67 (Thermo Fisher Scientific, RRID:AB_10853185 ), anti-cleaved caspase 3 (BD Pharmingen, RRID:AB_397274 ), anti-Brn2 (Santa Cruz Biotechnology, RRID:AB_2167385 ), anti-Ctip2 (Abcam, RRID:AB_2064130 ), and anti-GFP antibodies (Nacalai Tesque, RRID:AB_2313652 ; Medical and Biological Laboratories, RRID:AB_591819 ). After incubation with secondary antibodies and Hoechst 33342, the sections were washed and mounted.
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