Standard Hematoxilin & Eosin, Cresyl Violet and Luxol Fast Blue histochemical stainings were performed on paraffin sections and digitized using Mirax (Carl Zeiss) or Aperio (Leica) slide scanners as described previously [79 (link)]. Immunofluorescent detection was performed on free-floating sections as described [80 (link)] using the following antibodies: rabbit anti-ASPA serum (in house), mouse anti-NeuN (Merck), rat anti-MBP (Abcam), mouse anti-GFAP (Cell Signaling), chicken anti-β-Gal (Abcam), mouse anti-Flag (Cell Signaling) and rabbit anti-Neurofilament 200 (Sigma) followed by the appropriate Alexa-488/594 conjugated secondary antibodies (Thermo Fisher). Immunoperoxidase detection was performed on free-floating sections as described in [15 (link)] using mouse anti-APC (Merck) and rabbit anti-NeuN (Cell Signaling), the appropriate biotinolated secondary antibody (Dianova) and a Vectastain Elite ABC kit (Vector Labs). Immunoblotting was performed as described previously [78 (link)] with the following antibodies: rabbit anti-ASPA serum, mouse anti-GAPDH (Sigma), rat anti-MBP (Abcam), rat anti-PLP aa3 and rat anti-NG2 (gift of J. Trotter) followed by the appropriate HRP-conjugated secondary antibodies (Dianova). qRT-PCR for Aspa, Nat8l, NAAG synthetase-I and II (Rimklb and Rimkla) and hypoxanthine phosphoribosyltransferase (Hprt) was performed as described [26 (link)].
Rat anti mbp
Manufactured by Abcam
Sourced in United States, United Kingdom
Rat anti-MBP is a primary antibody that recognizes Myelin Basic Protein (MBP), a key structural protein found in the myelin sheath of neurons. This antibody can be used to detect and study MBP in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Roche (3 mentions)
Alexa 488, by Thermo Fisher Scientific (3 mentions)
Mouse anti gfap, by Merck Group (3 mentions)
Rabbit anti ng2, by Merck Group (3 mentions)
Rabbit anti iba1, by Fujifilm (3 mentions)
Mouse anti nestin, by BD (3 mentions)
Goat anti iba1, by Abcam (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Bradford assay, by Merck Group (2 mentions)
Rabbit anti pdgfrα, by Santa Cruz Biotechnology (2 mentions)
Anti rabbit krox20, by Abcam (2 mentions)
Goat anti rabbit and anti rat igg conjugated to horseradish peroxidase, by Merck Group (2 mentions)
Mouse anti cc1, by Abcam (2 mentions)
Mouse anti nestin, by Thermo Fisher Scientific (2 mentions)
Alexa 488 or alexa 594 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Mouse anti olig2, by Merck Group (2 mentions)
Mouse anti neun, by Merck Group (2 mentions)
Goat anti sox2, by Santa Cruz Biotechnology (2 mentions)
Alexa 546, by Thermo Fisher Scientific (2 mentions)
Mouse anti cdk2, by Santa Cruz Biotechnology (2 mentions)
21 protocols using rat anti mbp
1
Comprehensive Histochemical and Immunodetection Analysis
2
Quantitative Western Blot Analysis of MBP and Krox20
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Expression levels of MBP and Krox20 were quantitatively analyzed using western blot analysis. Samples were lysed in RIPA buffer (T&I) containing 1% SDS and protease inhibitor (aprotinin, leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride). The protein concentration of cell lysates was measured using the Bradford assay (Sigma). The samples were prepared in an SDS sample buffer and heated for 5 min at 95 °C; next, 10 μg of protein from each sample was loaded in SDS loading buffer and transferred to a polyvinylidene fluoride membrane. The membranes were blocked in 5% skim milk for 1 h, followed by incubation with anti-mouse Krox20 (1:1,000; BioLegend) and anti-rat MBP (1:500; Abcam) antibodies at 4 °C overnight. The membranes were washed three times in Tween-20 and incubated with goat anti-mouse IgG or anti-rat IgG conjugated to horseradish peroxidase (1:1,000; Sigma) for 2 h. Bands were visualized using an ECL system. The intensity of the blots was quantified with ImageJ software.
3
Immunostaining Analysis of Cell Markers
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Immunostaining analysis was performed as previously described (Yang et al., 2009 (link); Dincman et al., 2012 (link)). Anti-mouse A2B5 IgM and anti-mouse O4 IgM (50%, v/v) were produced by hybridoma culture. Anti-mouse Olig2 (1:1000), anti-mouse GFAP (1:1000), and anti-rabbit neurofilament (1:1000) were purchased from Millipore. Anti-rat MBP (1:500) was obtained from Abcam, anti-rabbit β-tubulin (1:1000) from Sigma, and anti-rabbit PDGFRα (1:500) from Santa Cruz. The Alexa-488 or Alexa-594 conjugated secondary antibodies were purchased from Invitrogen. The nucleic acid dye 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Roche. All quantitative data are presented as means ± SD. Statistical significance of the difference was evaluated by Student’s t-test. P < 0.05 was considered statistically significant.
4
Western Blot Analysis of Protein Expression
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The cells at co-culture system were lysed in RIPA buffer containing 1% SDS and protease inhibitor (aprotinin, leupeptin, pepstatin A, PMSF). The protein concentration of cell lysates was measured by using Bradford assay. The samples were prepared in an SDS sample buffer, heated for 3min at 98°C, and 6 μg of the protein from each sample were loaded in SDS loading buffer and then transferred to a PVDF membrane. The membranes were blocked in 2.5% skim milk for 1 h, followed by incubation with anti-rabbit Krox20 (1:1000, Abcam),anti-rat Sox 10 (1:800, Abcam) or anti-rat MBP (1:500, Abcam) antibodies at 4°C overnight. The membranes were washed three times in TBST and incubated with goat anti-rabbit and anti-rat IgG conjugated to horseradish peroxidase (1:1000, Sigma) for 2 h. Bands were visualized by using ECL system.
5
Quantitative Analysis of MBP and Krox20 Expression
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Expressions of MBP and Krox20 were quantitatively analyzed by using western blot analysis. The cells at DIV 1, 7, 14, and 21 were lysed in RIPA buffer containing 1% SDS and protease inhibitor (aprotinin, leupeptin, pepstatin A, PMSF). The protein concentration of cell lysates was measured by using Bradford assay. The samples were prepared in an SDS sample buffer, heated for 3 min at 98 °C, and 6 μg of the protein from each sample were loaded in SDS loading buffer and then transferred to a PVDF membrane. The membranes were blocked in 2.5% skim milk for 1 hr, followed by incubation with anti- rabbit Krox20 (1:1000, Abcam) or anti- rat MBP (1:500, Abcam) antibodies at 4 °C overnight. The membranes were washed three times in TBST and incubated with goat anti-rabbit and anti- rat IgG conjugated to horseradish peroxidase (1:1000, Sigma) for 2 hrs. Bands were visualized by using ECL system. The intensity of the blots was quantified with ImageJ software.
6
Immunochemical Analysis of Neural Cell Markers
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Immunochemical analysis was carried out as previously described (Yang et al. 2016 (link)). Anti-mouse O4 IgM (50%, v/v) were produced by hybridoma culture. Anti-mouse Olig2 (1:1000; Oasis Biofarm), anti-mouse GFAP (1:1000; Oasis Biofarm), and anti-rabbit neurofilament (NF-1) (1:1000) were purchased from Merck (Darmstadt, Germany). Anti-rat MBP (1:500), anti-rabbit EGFR (1:200) and anti-mouse CC1 (1:500) was obtained from Abcam (Boston, USA). Anti-mouse Nestin (1:1000) and the Alexa-488 or Alexa-594 conjugated secondary antibodies were purchased from Invitrogen (Frederick, USA). The nucleic acid dye 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Roche (Basel, Switzerland).
7
Quantitative Analysis of c-Jun and MBP in PNS Disease Model
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Expression levels of c-Jun and MBP were quantitatively analyzed using Western blot analysis. After PC film had been detached from the 3D PNS disease platform, all samples were lysed in radioimmunoprecipitation assay buffer (T&I, Chuncheon, Korea) containing protease inhibitors (aprotinin, leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride). The protein concentrations of the coculture lysates were measured using the Bradford assay (Sigma-Aldrich). Twelve to 15 μg of protein from each coculture sample were loaded in SDS–polyacrylamide gel electrophoresis gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with anti-rabbit c-Jun (1:500; Abcam) and anti-rat MBP (1:500; Abcam) at 4°C overnight. The membranes were washed three times in tris-buffered saline solution with Tween 20 and then incubated with goat anti-rabbit IgG or anti-rat IgG conjugated with horseradish peroxidase (1:1000; Sigma-Aldrich) for 2 hours. Bands were visualized using an ECL system, and the intensities of the bands were quantified using ImageJ software. In Figs. 3 (C and D) and 4 , each bar represents the average expression level of a protein of interest normalized to that of the control at DIV 17 and 28, respectively. Each protein level was normalized against β-actin, which was used as a loading control.
8
Immunochemical Analysis of Cell Markers
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Immunochemical analysis was carried out as previously described [22] . Anti-mouse O4 IgM (50%, v/v) were produced by hybridoma culture. Anti-mouse Olig2 (1:1000), anti-mouse GFAP (1:1000), and antirabbit neuro lament (NF-1) (1:1000) were purchased from Millpore. Anti-rat MBP (1:500) and anti-mouse CC1 (1:500) was obtained from Abcam, and anti-rabbit PDGFRα (1:500) from Santa Cruz. Anti-mouse Nestin (1:1000) and the Alexa-488 or Alexa-594 conjugated secondary antibodies were purchased from Invitrogen. The nucleic acid dye 4',6-diamidino-2-phenylindole (DAPI) was obtained from Roche.
9
Immunofluorescence Staining of Cells
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Cells were fixed in 4% paraformaldehyde (GIBCO) for 12 min and permeabilized with 0.1% Triton X-100 for 5 min. They were then blocked with 5% normal donkey or goat serum (Sigma) for 30 min and incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: Mouse anti-HA 1:400 (Sigma), Rabbit anti-HA (1:800), Chicken anti-NF200 (1:10,000), Rat anti-MBP 1:400 (Abcam), Rabbit anti-Brn3a 1:500 (Millipore), Phalloidin-TRITC conjugate 1:150 (Sigma). Cells were washed 3X with 0.1% PBS-TX before the species appropriate Alexa Fluorophore secondary antibodies were applied (all 1:1000). Coverslips were mounted with VECTASHIELD Antifade Mounting Medium (Vector Laboratories). Immunostaining was visualized using a confocal microscope (Zeiss LSM 700) and images were acquired using the Zen Black software.
10
Immunohistochemistry for Neural Cell Markers
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The following primary antibodies were used for immunohistochemistry: rabbit anti-active caspase-3 (1:500; Abcam, Cambridge, United Kingdom), rabbit anti-DCX (1:500; Cell Signaling Technology, Danvers, MA), rabbit anti-glial fibrillary acidic protein (GFAP) (1:500; Agilent, Santa Clara, CA), goat anti-Olig2 (1:50; R&D Systems, Minneapolis, MN), rabbit anti-Iba1 (1:500; Fuji-Wako, Osaka, Japan), rabbit anti-Ki67 (1:500; Abcam), goat anti-MBP (1:500; Santa Cruz Biotechnology, Dallas, TX), rat anti-MBP (1:1,000; Abcam), mouse anti-NeuN (1:100; Merck-Millipore, Burlington, MA), and rat anti-NG2 (1:500; Abcam). The conjugated secondary antibodies were donkey anti-rabbit, anti-rat, anti-mouse, and anti-goat antibodies (Alexa Fluor 488, 568, and 647; Thermo Fisher Scientific, Waltham, MA) at a dilution of 1:500. For the EdU reaction, azide-modified dyes (Alexa Fluor 488 and 594 azide; Thermo Fisher Scientific) were used.
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