Hello, Mohamed:

I think that it's not related to processing or cutting because when I made an immuno without using Nissl staining there are no cracks in sections, so I don't know if dehydrated steps are too strong.

Hi.. Actually I too think it is from cutting and tissue processing. Or prior to embedding, the dehydration steps are too harsh. If you may share the protocol and what type of fixation and embedding you use, I will help you with it.

The protocol is the following:

1. Hydration:

EtOH 100 II (30 sec)

EtOH 100 I (30 sec)

EtOH 90 II (30 sec)

EtOH 90 I (30 sec)

EtOH 70 I (1 minute)

Nissl (2 minutes)

H2O distilled (20 sec)

2. Dehydration:

EtOH 70 I (30 sec)

EtOH 90 I (30 sec)

EtOH 90 II (30 sec)

EtOH 100 I (30 sec)

EtOH 100 II (1 minute)

Histoclear (at least for 2 hours)

Mounting: using Pertex or Entellan.

Thanks Alejandro. This protocol appears fine. What about before embedding? dehydration steps before the embedding actually takes place may render the tissue brittle and susceptible to cracks. Or if tissue clearing is done in xylene, keeping it for longer than 15-20 min in xylene usually renders brittleness to the tissue. We keep it in cedar wood oil for clearing. I do not know whether it can be done effectively in brain sections. But you may try.

Is your tissue sectioning done in microtome or cryotome? As paraffin embedding has its own advantages and disadvantages. I believe something may be going wrong before the tissue comes on slide.

However, what surprises me is that you also mentioned that your immunohistochemistry on the same sections did not have such cracks! And your protocol here does not at all indicate a harsh dehydration. On the other hand, I dip the sections for 3-10 min (depending on the tissue type) in each grade of alcohol.

Should defatting of your tissue be done using xylene and ethanol (10-15 min)? Since brain has a very high lipid content.

We're using free-floating sections for immunohistochemistry. The zebrafish brains were extracted and postfixed for 48 hours in 4% paraformaldehyde, cryoprotected in 30% sucrose, and then cut in 30 um sections using a microtome equipped with a freezing stage.

Okay. What about these conditions when using the sections for Nissl's staining?

The conditions are the same for both immunostaining and immunostaining + Nissl's staining. The cracks remain in both cases.

Could it be that Nissl reveals cracks that you can't see with immuno (but are still there)? In which case, maybe your cutting temperature could be too low. I've found that this can give cracks to ammniote brain tissue.

In Bilken university, in Turkey, about 5 years ago, they worked on the brain of zebrafish. Maybe you can search for the team and have contract with them. best regards.