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Anti-O4, clone 81 (also referred to in the literature as mAB O4)
| Species Reactivity | Key Applications | Host | Format | Antibody Type |
|---|---|---|---|---|
| Ch, H, M, R | IC, IH, not IP, not WB | Mouse | Purified | Monoclonal Antibody |
Description:
Anti-O4, clone 81 (also referred to in the literature as mAB O4)
Trade Name:
Chemicon (Millipore)
Specificity:
Recognizes Oligodendrocyte marker O4. Also reacts with certain galactolipids in sperm (see Additional Information library for list).
Immunogen:
Homogenate of white matter of corpus callosum from bovine brain.
Clone:
81 (also referred to in the literature as mAB O4)
Isotype:
IgM
Background Information:
Oligodendrocytes and astrocytes are derived from common precursor cells, glioblasts (Sommer, 1981). O-Antigens are sulfatides, which function as differentiation markers on the surface of oligodendrocytes of the central nervous system. O4 is formed postnatally (Schachner, 1981) and is a marker for cell bodies and processes of oligodendrocytes types I and II (hairy eyeball type). During oligodendrocyte differentiation, O4 occurs in pro-oligodendrocytes, but not in O-2A-progenitor cells. O4 occurs from day 3 onwards in cell cultures of embryonic mouse brain. Anti-O4 can be used in myelination, demyelination and remyelination studies and in regeneration experiments.
Species Reactivity:
- Chicken
- Human
- Mouse
- Rat
Application Notes:
Immunohistochemistry: 10-20 μg/mL on unfixed, shock frozen tissue.
Immunocytochemistry: 10-20 μg/mL on cells fixed with 4% paraformaldehyde.
Note: O4 is a sulfatide, which can be dissolved out of the membrane by organic solvents; acetone and methanol should not be used for fixation.
Optimal working dilutions must be determined by the end user.
Immunohistochemistry protocol
1. Prepare sections from unfixed, shock frozen tissue. The sections should be 4-5 μm thick. Place the sections on microscope slides.
2. Wash the slide three times for 5 min. each in PBS at room temperature.
3. Block the non-specific binding sites by incubating the sections in a humid chamber with 5% FCS at room temperature for 30 minutes.
4. Wash the slides as described in step 2.
5. Cover the sections with a sufficient amount of MAB345 (10-20 μg/mL in PBS) and incubate in a humid chamber at 37°C for one hour.
6. Wash the slides briefly three times with PBS. Carefully dry around the area to be stained.
7. Cover the sections with a sufficient amount of anti-mouse IgM-fluorescein* solution and incubate in a humid chamber at 37°C for one hour.
8. Wash the slides as described in step 6.
9. Cover the sections with a suitable embedding medium, cover with a cover slip, and examine by fluorescence microscopy.
*HRP or ABC can also be used.
Optimal results can be obtained by titrating the primary and secondary antibodies
Immunocytochemistry
1. Fix the preparations with 4% paraformaldehyde (in PBS) at room temperature for 10 minutes. O4 is a sulfatide which can be dissolved out of the membrane by organic solvents; acetone and methanol should not be used for fixation.
2. Wash the slide three times for 5 min. each in PBS at room temperature.
3. Block the non-specific binding sites by incubating the sections in a human chamber with 5% FCS at room temperature for 30 minutes.
4. Wash the slides as described in step 2.
5. Cover the sections with a sufficient amount of MAB345 (10-20 μg/mL in PBS) and incubate in a humid chamber at 37°C for one hour.
6. Wash the slides briefly three times with PBS. Carefully dry around the area to be stained.
7. Cover the sections with a sufficient amount of anti-mouse IgM-fluorescein solution and incubate in a humid chamber at 37°C for one hour.
8. Wash the slides as described in step 6.
9. Cover the sections with a suitable embedding medium, cover with a cover slip, and examine by fluorescence microscopy.
Note: Do not allow the preparations to dry out during staining.
Immunocytochemistry: 10-20 μg/mL on cells fixed with 4% paraformaldehyde.
Note: O4 is a sulfatide, which can be dissolved out of the membrane by organic solvents; acetone and methanol should not be used for fixation.
Optimal working dilutions must be determined by the end user.
Immunohistochemistry protocol
1. Prepare sections from unfixed, shock frozen tissue. The sections should be 4-5 μm thick. Place the sections on microscope slides.
2. Wash the slide three times for 5 min. each in PBS at room temperature.
3. Block the non-specific binding sites by incubating the sections in a humid chamber with 5% FCS at room temperature for 30 minutes.
4. Wash the slides as described in step 2.
5. Cover the sections with a sufficient amount of MAB345 (10-20 μg/mL in PBS) and incubate in a humid chamber at 37°C for one hour.
6. Wash the slides briefly three times with PBS. Carefully dry around the area to be stained.
7. Cover the sections with a sufficient amount of anti-mouse IgM-fluorescein* solution and incubate in a humid chamber at 37°C for one hour.
8. Wash the slides as described in step 6.
9. Cover the sections with a suitable embedding medium, cover with a cover slip, and examine by fluorescence microscopy.
*HRP or ABC can also be used.
Optimal results can be obtained by titrating the primary and secondary antibodies
Immunocytochemistry
1. Fix the preparations with 4% paraformaldehyde (in PBS) at room temperature for 10 minutes. O4 is a sulfatide which can be dissolved out of the membrane by organic solvents; acetone and methanol should not be used for fixation.
2. Wash the slide three times for 5 min. each in PBS at room temperature.
3. Block the non-specific binding sites by incubating the sections in a human chamber with 5% FCS at room temperature for 30 minutes.
4. Wash the slides as described in step 2.
5. Cover the sections with a sufficient amount of MAB345 (10-20 μg/mL in PBS) and incubate in a humid chamber at 37°C for one hour.
6. Wash the slides briefly three times with PBS. Carefully dry around the area to be stained.
7. Cover the sections with a sufficient amount of anti-mouse IgM-fluorescein solution and incubate in a humid chamber at 37°C for one hour.
8. Wash the slides as described in step 6.
9. Cover the sections with a suitable embedding medium, cover with a cover slip, and examine by fluorescence microscopy.
Note: Do not allow the preparations to dry out during staining.
Applications Not Recommended:
- Immunoprecipitation
- Western Blotting
Control:
Rat cortical stem cells or day 3 cell cultures of brains from mouse embryos
Purification Method:
Ammonium sulfate precipitation
Presentation:
Purified immunoglobulin in 0.05M Potassium phosphate buffer, pH 8.0 with 0.3M NaCl and 0.05% sodium azide.
Storage Conditions:
Maintain for 1 year at 2–8°C from date of shipment. Aliquot to avoid repeated freezing and thawing. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Alternate Names:
Sulfatide
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Key Applications:
- Immunocytochemistry
- Immunohistochemistry
Qty/Pk:
50 μg
Brand Family:
Chemicon
Format:
Purified
Host:
Mouse
Product Name:
Anti-O4, clone 81 (also referred to in the literature as mAB O4)
Concentration:
1.05 mg/mL
Antibody Type:
Monoclonal Antibody