Tissues were permeabilized with TBS-T (TBS with 0.1% Tween-20) and blocked with 5% bovine serum albumin (BSA, Sigma) for 1 h. After blocking, tissues were incubated overnight 4°C with primary antibodies (Mouse anti-MBP 1:520, sc-271524 Santa Cruz; Rabbit anti-Neurofilament 1:750, ab1987 Millipore, United States; Rabbit anti-MANF 1:1,000, LS-B2688, LSBio, United States; Goat anti-TPPP, 1:500, PA5-19243, Invitrogen, United States) diluted in blocking solution. The next day, tissues were washed three times with TBS-T, and incubated for 1 h with fluorescence-conjugated corresponding secondary antibodies (Alexa 488, 568, and 647, 1:200, donkey anti-rabbit, anti-goat, and anti-mouse, Thermo Fisher Scientific), diluted in blocking solution at room temperature. Tissues were washed and incubated with DAPI to visualize nuclear and counterstain (1:1,000, Thermo Fisher Scientific). Laser scanning confocal micrographs of the fluorescently labeled lumbar spinal cords were acquired with an LSM700 (Carl Zeiss) using a LCI Plan-Neofluar 63×/1.3 Imm Corr objective with 80% glycerol.
Mouse anti mbp
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse anti-MBP is an antibody that specifically binds to the myelin basic protein (MBP) found in the central nervous system. It is used in research applications to detect and study MBP expression and distribution.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Sc 271524, by Santa Cruz Biotechnology (1 mentions)
Mouse anti sox10, by Santa Cruz Biotechnology (1 mentions)
Phalloidin ifluor 488 reagent, by Abcam (1 mentions)
Mn2 phos tag sds page, by Fujifilm (1 mentions)
Image pro plus software 5, by Media Cybernetics (1 mentions)
Mouse anti olig2, by Merck Group (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (1 mentions)
Ab8898, by Abcam (1 mentions)
5 protocols using mouse anti mbp
1
Immunohistochemical Profiling of Spinal Cord
2
Immunostaining of Sox10 and MBP in Cells
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Cells fixed in 4% PFA at RT for 30 minutes were permeabilized with 0.1% triton X‐100 in PBS for 5 minutes after washing with 1× PBS. Then, the cells were washed 2 to 3 times and blocked in PBS with 1% BSA at RT for 1 hour. The cells were incubated with primary antibodies: mouse anti‐Sox10 (sc‐365 692, Santa Cruz, 1:200), mouse anti‐MBP (sc‐271 524, Santa Cruz, 1:200) antibodies at 4°C overnight in blocking solution. After washing with 1x PBS, the cells were incubated with Alexa Flour 594‐conjugated secondary antibodies (mouse A11005, Invitrogen, 1:500) or Phalloidin‐iFluor 488 reagent (ab176753, Abcam) at RT for 1 h. For generating imaging data, randomly selected fields (≥3) were photographed using a confocal microscope (Carl Zeiss, LSM 700, Germany) and analyzed using ImageJ software (NIH) in all of imaging data.
3
In Vitro Phosphorylation of Lgl by aPKC
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A reaction mixture containing 15 μM MBP-Lgl
647–673, the aPKC kinase domain, and 1 mM ATP in the reaction
buffer described above was set up and incubated at 30 °C for
90 min. Small aliquots of the reaction mixture were quenched via sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)
loading buffer periodically during the incubation. The quenched samples
were run on Mn2+-Phos-tag SDS–PAGE (Wako USA). The
phosphorylated Lgl was detected on a Western blot using mouse anti-MBP
(1:1000, Santa Cruz Biotechnology) and bovine anti-mouse HRP (1:2000,
Santa Cruz Biotechnology) antibodies.
647–673, the aPKC kinase domain, and 1 mM ATP in the reaction
buffer described above was set up and incubated at 30 °C for
90 min. Small aliquots of the reaction mixture were quenched via sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)
loading buffer periodically during the incubation. The quenched samples
were run on Mn2+-Phos-tag SDS–PAGE (Wako USA). The
phosphorylated Lgl was detected on a Western blot using mouse anti-MBP
(1:1000, Santa Cruz Biotechnology) and bovine anti-mouse HRP (1:2000,
Santa Cruz Biotechnology) antibodies.
4
Quantification of Myelin Proteins in Olig2 Mutant Mice
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At the age of P21, mice (n = 7 in WT, n = 8 in Olig2 cKO) were anesthetized with 1% pentobarbital. Cerebral cortex (anterior cingulated area) and corpus callosum were rapidly removed and frozen. Frozen samples were homogenized and proteins were extracted using RIPA lyses buffer with protease inhibitors (Roche). Lysates containing 40 μg protein were denatured in gel-loading buffer, separated on 10% SDS-PAGE gels, transferred to PVDF membranes and visualized by chemiluminescence (ECL plus, GE Healthcare). Quantification of band intensity was analyzed using Image-Pro Plus software 5.0 (Media Cybernetics). The following primary antibodies were used: mouse anti-Olig2 (1:500; Millipore), mouse anti-MBP (1:1000; Santa Cruz Biotechnology) and mouse anti-β-actin (1:2000; Santa Cruz Biotechnology).
5
Immunohistochemical Analysis of Myelin and Histone Modifications
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Mice for each group were deeply anesthetized with 1% sodium pentobarbital and transcardially perfused with 4% paraformaldehyde in PBS. Brains were dehydrated in 10, 20, and 30% sucrose in 4% paraformaldehyde for 12 hours, respectively. The frozen brains were sectioned (20 μm) on acryostat microtome (MS1900, Leica). Immunostaining was performed as previously described (20 (link)). Free-floating sections were incubated with primary antibodies overnight at 4°C after 1 h blocking with 5% BSA, followed by secondary antibodies incubation (1.5 h). Primary antibodies included: mouse anti-APC (1:500, OP-80, Millipore), rabbit anti-H3K9me3 (1:500, ab8898, Abcam), and mouse anti-MBP (1:200; Santa Cruz). Alexa fluor 488-conjugated secondary antibodies against mouse and 568-conjugated secondary antibodies against rabbit (1:2000; Invitrogen) were used.
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