paraffin sections of mouse brain

[nesugi]

I am doing immunostaining of 4% praformaldehyde (PFA) perfusion fixed and paraffin embedded mouse brain sections.
The results showed a lot of vacuoles where neuronal cells should be present.
Is this due to too much dehydration of brain blocks befor the paraffin embedding?
I put 4% PFA fixed brain blocks in ethanol overnight for dehydration?
Or is there any other reasons?
I would appreciate if you give me an advice.

[excalibur]

After perfusion, are you further fixing the brain? Perfusion is usually not enough for thorough fixation.

You should gradually dehydrate tissue in alcohol, starting with 70%, then 95%, then 100%. A couple changes of each.

What clearant are you using between alcohol and paraffin?

[nesugi]

Thank you very much for replying.

1)Yes, I performed post fixation of perfused brain in 4% PFA for 32 hrs.

2)Yes, I gradually dehydrate tissue in alcohol, starting with 70%, then 95%.  Finally I kept tissue in 100% alcohol overnight.

3)I used chloroform as a clearant.

[excalibur]

Well, everything sounds good. Chloroform is great, because it is less drying than xylene and my preferred clearant for eyes.

Have you stained any sections with just H&E? If so, what do they look like?

Make sure the temp of your paraffins never exceeds 60C. I even keep mine around 58C.

[nesugi]

Thanks again.
I am keeping paraffins at 60-62C.
I will try H&E staining from now.

[richard03]

I would never leave my tissues in 100% alcohol for that long. You should always leave tissues in 70% alcohol for longer storage.

I kept tissue in 100% alcohol overnight.