Analysis Note

ControlRat cortical stem cells or day 3 cell cultures of brains from mouse embryos

Application

Anti-O4 Antibody, clone 81 is an antibody against O4 for use in IC, IH with more than 50 product citations.

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.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

Concentration: Varies, see lot specific CoA

Physical form

Purified immunoglobulin in 0.05M Potassium phosphate buffer, pH 8.0 with 0.3M NaCl and 0.05% sodium azide.

Format: Purified

Specificity

Recognizes Oligodendrocyte marker O4. Also reacts with certain galactolipids in sperm (see Additional Information library for list).