The expression of MTR, BHMT, LC3B, and P62 in the ovaries was detected using IHC. The sections were baked at 65°C in an oven, dehydrated with alcohol, incubated with 3% H2O2, boiled in 10 mM citrate buffer (pH 6.0) for antigen retrieval, rinsed in Tris-buffered saline (TBS) three times, added to solution A (for biotin block), and washed three more times in TBS. Sections were incubated in goat serum for 1 h at room temperature, and were then incubated separately with anti-MTR (1:200, ab66039, Abcam, UK), anti-BHMT (1:200, ab243698, Abcam, UK), anti-LC3B (1:300, ab48394, Abcam, UK), and anti-P62 (1:200, F032602, Abways Technology) overnight at 4°C. Samples were then washed in PBS. Sections were incubated with a goat anti-rabbit secondary antibody at 37°C for 30 min, and the sections were then stained with 3,3'-diaminobenzidine and counterstained with hematoxylin at room temperature. The negative control sections were incubated in biotinylated goat antirabbit immunoglobulin G (1:200 dilution) and stained with the experimental sections. Densitometric quantification was performed using ImageJ software.
Ab66039
Manufactured by Abcam
Sourced in United Kingdom
Ab66039 is a laboratory equipment product manufactured by Abcam. It is designed to perform a specific function within a research or analytical setting. The core function of this product is to enable users to carry out their work efficiently and accurately. No further details about the intended use or performance of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab243698, by Abcam (2 mentions)
Ab48394, by Abcam (2 mentions)
F032602, by Abways (2 mentions)
Ab0037, by Abways (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Coolpix 4300, by Nikon (1 mentions)
Immuno blot pvdf, by Bio-Rad (1 mentions)
Trans blot sd semi dry electrophoretic transfer cell, by Bio-Rad (1 mentions)
Gapdh, by Bio-Rad (1 mentions)
5 protocols using ab66039
1
Ovarian Expression of Metabolic Markers
2
Western Blot Analysis of Ovarian Proteins
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Lysates from ovarian tissue and mGCs were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were incubated with MTR antibody (1:2000, ab66039, Abcam, UK), BHMT antibody (1:200, ab243698, Abcam, UK), LC3B antibody (1:1000, ab48394, Abcam, UK), P62 antibody (1:1000, F032602, Abways Technology), Beclin-1 antibody (D40C5, 1:500, 3495s, Cell Signaling Technology, Inc.), mTOR (s2442) antibody (1:1000, BS3611, bioWORLD), and p-mTOR antibody (1:1000, BS4706, bioWORLD). In addition, samples were incubated with peroxidase-conjugated secondary antibody, and an enhanced chemiluminescence detection kit (Amersham Pharmacia Biosciences, Inc.) was used for visualization. Protein levels were quantified and normalized using glyceraldehyde 3-phosphate dehydrogenase (1:10,000, AB0037, Abways Technology) as internal control.
3
Immunohistochemical Analysis of MTR Expression
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The expression of MTR was also confirmed by immunohistochemistry. Sections were baked at 65°C, dehydrated, and incubated in 3% H2O2. Sections were subjected to antigen retrieval by boiling in 10 mM citrate buffer (pH 6.0), and rinsed in TBS. We added solution A (Biotin block), washed with TBS, added solution B (Biotin block), and we again washed sections with TBS. Sections were incubated in normal goat serum for 1 h at 4°C, incubated with anti-MTR (1:200, ab66039, Abcam, UK) in 3% BSA overnight at 4°C, incubated in biotinylated goat anti-rabbit IgG (1:200 dilution) for 2 h at room temperature, and then washed with PBS. We added reagent SABC (12E02A, BOSTER, Wuhan, China) and incubated sections at 37°C for 20 min, washed with PBST, visualized binding with DAB, and terminated the incubation with distilled water. Slides were stained with hematoxylin, dehydrated, and mounted. The average integrated optical density (IOD) was measured for each sample by Image-pro Plus 6.0 (Media Cybernetics, USA).
4
Immunoblotting Analysis of Cobalamin-Dependent Proteins
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Worms grown in the absence of CN-Cbl or in the presence of CN-Cbl or the CN-Cbl dodecylamine derivative were homogenized in 100 mmol/L potassium phosphate buffer (pH 7.0) at 4 °C. Each homogenate was centrifuged at 15,000×g for 10 min and the supernatant fraction was analyzed. We used a precast slab gel (PAGEL, type NPG-520L, ATTO Corporation, Tokyo, Japan) for electrophoresis of samples through a 5–20% (w/w) linear gradient of polyacrylamide in the presence of SDS. After electrophoresis, proteins were transferred to a PVDF membrane (Immuno-Blot PVDF, Bio-Rad Laboratories Inc., Hercules, CA, USA) in a Trans-Blot SD semi-dry electrophoretic transfer cell (Bio-Rad). The PVDF membrane was probed with an anti-MS antibody (ab66039, abcam®, Cambridge, MA, USA). We performed the immunodetection reactions using an anti-mouse IgG antibody secondary antibody (Promega KK, Tokyo, Japan) coupled to horseradish peroxidase and an immunoblot-staining kit for peroxidase (EzWestBlue, ATTO), according to the manufacturer’s instructions. A Protein Ladder One Triple-color kit (Nacalai Tesque Inc., Kyoto, Japan) was used to determine molecular mass. After the treated PVDF membrane was photographed using a digital camera (Coolpix 4300, Nikon, Japan), the intensities of the protein bands were calculated of Image J software [24] .
5
Ovarian Protein Expression Analysis
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Ovarian lysates from mice were separated by SDS/PAGE and transferred onto nitrocellulose membranes. Membranes were probed with polyclonal MTR antibodies (1:2000, ab66039, Abcam, UK), and blots were visualized by using peroxidase-conjugated second antibody and an ECL detection kit (Amersham Pharmacia Biosciences). Western blot data were quantified and normalized to GAPDH (1:10000, Bio-Rad, USA).
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