Proteins extracts were subjected to polyacrylamide gel electrophoresis and electrotransfer to PVDF membranes. The membranes were blocked with 5% non-fat milk and then probed with the indicated antibodies in TBS-Tween, 5% (w/v) non-fat dry milk overnight at 4°C. Antibodies used were against 2H8 anti-turboGFP (Origene, TA150041, 1:2000), anti-turboRFP clone OTI2D9 (Origene, TA150061, 1:1000), anti-Gapdh (Sigma, G9545, 1:2000), anti-RARA(c-20) (Santa Cruz, sc-551, 1:50), anti-RARB(C-19) (Santa Cruz, sc-552, 1:50), anti-RARG(C-19) (Santa Cruz, sc-550; 1:50), and β-tubulin (Sigma, T3952, 1:2000).
T3952
Manufactured by Merck Group
The T3952 is a laboratory centrifuge designed for general-purpose sample separation and processing. It provides consistent and reliable performance for a variety of applications. The core function of this equipment is to separate different components of a liquid mixture by applying centrifugal force.
Automatically generated - may contain errors
Lab products found in correlation
β tubulin, by Merck Group (1 mentions)
Ta150041, by OriGene (1 mentions)
Hrp linked secondary antibody, by Cell Signaling Technology (1 mentions)
Ab6320, by Abcam (1 mentions)
Ab75485, by Abcam (1 mentions)
Anti lamin a c, by Merck Group (1 mentions)
Goat anti rabbit hrp, by Agilent Technologies (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Phospho akt s473, by Cell Signaling Technology (1 mentions)
Dmi4000b microscope, by Leica (1 mentions)
Cm1860, by Leica (1 mentions)
Mouse anti cc1, by Merck Group (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Mouse anti brn3a, by Santa Cruz Biotechnology (1 mentions)
Mab377, by Merck Group (1 mentions)
Ab144p, by Merck Group (1 mentions)
Alexa fluor conjugated secondary antibodies, by Biotium (1 mentions)
Z0334, by Agilent Technologies (1 mentions)
L9393, by Merck Group (1 mentions)
5 protocols using t3952
1
Immunoblotting Analysis of Protein Targets
2
Irradiation and Pharmacological Modulation of Stemness
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Cells were first irradiated with 4 Gy and then treated with 10 μM RO4929097, 10 μM TMZ as well as their combination. 4 days post-treatment total cell lysate was prepared using SDS lysis buffer. Lysates were blotted onto a PVDF-membrane. Membranes were probed overnight at 4°C with primary antibodies and bound antibodies were visualized using HRP-linked secondary antibodies (Cell-Signaling) and ECL Luminescence (Pierce Biotechnology). Anti-SOX2 (ab75485, Abcam), anti-Nestin (ab6320, Abcam), anti-beta-tubulin III (T3952, Sigma-Aldrich), and anti-Lamin A/C (Sigma-Aldrich) were used at 1:1000 dilution.
3
Protein Extraction and Western Blot Analysis
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Mouse brains were homogenized in RIPA buffer supplemented with sodium fluoride (2 mM), sodium orthovanadate (1 mM), sodium pyrophosphate (1 mM), β-glycerophosphate (2.5 mM), PMSF (1 mM), DTT (1 mM) and protein inhibitor cocktail (Roche; Basel, Switzerland). The primary antibodies that were used in this study are phospho-AktS473 (1:1000; D9E, #4060; Cell Signaling Technology; Danvers, MA), total Akt (1:1000; #9272; Cell Signaling Technology), phospho-Erk1T202/204/Erk2T183/Y185 (1:1000; sc-16982; Santa Cruz Biotechnology, Dallas, TX), total Erk1/2 (1:1000; C16, sc-93; Santa Cruz Biotechnology) and β–tubulin (1:1000; T3952; Sigma-Aldrich, St. Louis, MO). Goat-anti-rabbit-HRP (1:2000; DAKO, Santa Clara, CA) was used a secondary antibody.
4
Immunostaining of Retina and Optic Nerve
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Retina and optic nerves were fixed with 4% PFA for 2 h at RT. Blocking solution was made with PBS including 10% normal goat serum and 0.3% TX-100. The primary and secondary antibodies were diluted in blocking solution and incubated at 4°C for overnight. Antibodies include mouse anti-Brn3a (1:100, Santa Cruz, Sc-8429), rabbit anti-Iba1 (1:1,000, Wako, 019–19741), mouse anti-CC1 (1:500, Millipore Sigma-Aldrich, OP80), and mouse (BioLegend, 801202) or rabbit (Millipore Sigma-Aldrich, T3952) anti-TUJ1 (1:1,000). The following day, Alexa Fluor–conjugated secondary antibodies were incubated for 2 h at RT (Table S1 ). Tissue samples were mounted in Vectashield (with DAPI) for visualization with a Leica DMI4000B microscope under confocal settings. For the 16-µm tissue sections of retina or optic nerve (Fig. S1 ), a cryostat (Leica CM1860) was used.
5
Spinal Cord Tissue Preparation and Staining
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Anesthetized mice were transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4. Spinal cords were dissected and postfixed overnight in the same fixative at 4 • C. The fixed tissues were cryoprotected successively in 10 % and 20 % sucrose in PBS overnight at 4 • C, embedded in OCT compound, and sectioned axially or sagittally at 50 m or 20 m on a cryostat, respectively. Tissue sections were stained with primary antibodies to glial fibrillary acidic protein (GFAP) (1:500, Z0334, DAKO, Carpinteria, CA, RRID: AB 10013382), Laminin (1:500, L9393, Sigma, St. Louis, MO, RRID: AB 477163), 5-hydroxytryptamine (5-HT) (1:200, 1338, ImmunoStar, Hudson, WI, RRID: AB 572263), NeuN (1:500, MAB377, Millipore, Billerica, MA, RRID: AB 2298772), ChAT (1:500, AB144 P, Sigma, St. Louis, MO, RRID: AB 2079751), -tubulin III (1:500, T3952, Sigma, St. Louis, MO, RRID: AB 1841226). The sections were then incubated with Alexa Fluor-conjugated secondary antibodies (1:500, Biotium, Fremont, CA). Nuclei were stained with Hoechst (bisbenzimide H33258 fluorochrome trihydrochloride, Nacalai Tesque).
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