Hello everyone,

I am having trouble in myelin protein staining in mouse spinal cord section. I am following the protocol as following;

1- Anesthetize the mouse using isoflurane

2- Cardiac perfusion with 4% PFA

3- Collecting spinal cord (still in vertebrae)

4- After 48-72h, remove the spinal cord from vertebrae and transfer in freshly prepared 30% sucrose (in PBS) solution for cryoprotection

5- Once tissue settle down (>2 days), dissect out spinal cord as per cervical/thoracic/lumbar regions.

6- Prepare OCT block and cut 14u thick section on microtome

7- Store the slides in -80 C freezer

For staining,

1- Take out the slide from freezer and warm at room temp for 1 hr.

2- Rehydrate the section in PBS and start staining

3- Perform heat antigen retrieval in 10mM citrate buffer (pH-6)

4- After washing in PBS, treating with 3% H2O2/2% triton X 100 for 30 min

5- Post PBS washing, blocking in 5% NGS/0.3% triton X 100, 1h RT

6- Incubating in primary antibody (PLP, 1:250) overnight at 4 C or RT

7- Next day after washing, incubating with 20 antibody (1:500), 1h, RT

8- Post washing, Vector lab ABC treatment (1h, RT)

9- Developing the reaction using DAB method and mounting

The problem is that when we look under microscope, white matter area staining looks lighter than cortex, that is completely weird. We observed same problem in fluorescent staining.

I will appreciate if someone could pinpoint the issue and help in troubleshooting this problem.