Dear all,

have an antibody that stains the surfaces of my brain slices strongly, but the center is weakly stained. Please see the attached PDF for a comparison of three stainings (scanned in X-Z-mode). The bottom staining is the one that needs improvement.

Since we would like to quantify the staining in optical sections throughout the whole slice, I need to improve the labeling.

General protocol:

• Mouse brain fixed in 4% formalin overnight
• 50 µm thick vibratome-sections
• Free-floating staining
• No retrieval
• Blocking for 60 Minutes in PBS with 5% NDS + 0,3%Tx-100
• Primary AB 1:1000 in PBS + 3% NDS+0,3% Tx-100 over night at 4°C
• Detection with Donkey anti Primary 1:500 in PBS + 3% NDS (60-120 Minutes incubation at RT)

Modifications I have tried (that did not solve the issue):

• Longer incubation time
• Antigen retrieval (citrate-based)
• Dilution series from 1:250-1:64000

As you can see in the PDF, one can see this effect in staining 2, too. Both stainings 2&3 have in common, that the labeled protein is located in the membrane. I do not observe this issue in cytosolic proteins.

Unfortunately, my analysis requires a certain section thickness and the ability to analyze more than just the surface of the slice.

I would be grateful for ideas about how to fix this issue.

Best regards

Henrike

Edit for clarification: I replaced "intracellular proteins" by "cytosolic proteins" (PDF not updated). All three epitopes are located intracellularly.