HI,

Immediate thing which comes to my mind is the pH of the saline. Pls check the pH. 

We generally perfuse animals transcardially with 4% paraformaldehyde in 0.1 M phosphate buffered saline, pH 7.2, and the tissues were post-fixed after with 4% PFA again.

You can post-fix using 4%PFA instead of formalin and check it.. Hope it helps.

It is a perfusion issue, which needs to be improved/fixed. can be fixed. Good luck!

Dear Pandi-Perumal, 

Can you please help me which step am i going wrong??  Or How can i fix the perfusion issue ??? Please help

Over the years, I have worked in the wet labs. However, I have moved to clinical field. You may try the method proposed by Santosh. A decade ago, I was interested in looking at some specific brain cells with a particular staining. I requested some brain specimen from overseas. The PI was a busy person. He requested one of his junior associate (as the expert has gone on leave) to perfuse and ship the brain specimen. The fact that the perfusion was not done correctly, we had problem fixing, transferring, and sectioning. When we looked the mounted slides, the sections were too bad. The tissues come off easily in every procedure. We end up discarding the entire shipment due to bad quality of perfusion. I thought I could tell you that there are issues such as this one, which you need to overcome!

As I am not an expert on perfusion, you could wait for others to give their inputs. Good luck!!! 

if you are fixing using PFA by perfusion, why dont you perform cyropreservation and cryosectioning

Hi Ray!!!

 I think that could be because of not proper fixation, tissue might have putrefied. the problem may be with perfusion or may be PFA or preparation of PFA. 

I see two major possible reasons of your problem:

1) As mentioned by Santosh, check your PFA solution pH. I normally dissolve PFA in standart phosphate buffered saline and the pH is around 7.1-7.2.

2) Try another stock of dry PFA - yours may be not pure enough or just contaminated.

Also try to fix the braain in the same 4% PFA solution instead of 10% formalin.

Good luck!

Hello,

This is a tissue fixation and/or tissue processing issue.  The brain tissue was perfused and placed in either pH balanced 4% paraformaldehyde or 10% neutral buffered formalin(NBF) for fixation.  As fixation is critical for tissue preservation, allow the fixative to penetrate at a rate of 1mm per hour.  The fixation of your animal brains for 2-3 days in 10% formalin might be sufficient. However, different animals require longer periods in a fixative. You should visualize the tissue for consistency and uniformity of fixation prior to proceeding to the next step. 

The brain must go through a tissue processor prior to tissue embedding in paraffin.  The tissue processor will complete cycles of dehydration with an increasing concentration of alcohols, clearing with xylene or substitute, and infiltration with paraffin.  According to your steps above, you went from tissue fixation directly to tissue embedding.  Then during microtomy you noticed the sunken regions of the hippocampus. If so, the brain tissue did not complete the tissue processing step. 

The sunken region of the hippocampus may not only be a fixation problem but also tissue processing problem. You can not go directly from fixation to embedding.  The hydration of the fixative will not allow the paraffin to properly infiltrate the tissue.  Your problem can be prevented by following the steps to create tissue slides from paraffin blocks.

Fixation-tissue processing-embedding-microtomy-staining-coverslipping 

Hope this helps and Good Luck,

Brian Johnson

Hi Ray Radha Sargunam 

Fixation, dehydration and clearing have to be done with caution to get good results in histology. The more you fix the more the brain (or other tissues) shrunk and harden. That's why it seems wise to reduce fixative concentration. 4% PFA is more than enough also during postfixation, which can be even shortened to overnight if you do a good perfusion. If you are using rats I would recommend perfusing with at least 400 mL, at a rate of 20 mL/minute. If you are using mice, 60 mL, 5-6 mL/min. This gives better results that a very short perfusion with a long postfixation.

After fixation it is very important to rinse the brain to eliminate completely any trace of the fixative. This can be done by rinses of the tissue piece many times with saline or PBS in a rocking table. Depending on your purpose I would even recommend cleaning for many hours in running tap water. But if you plan to do fine histology (e.g, immunocytochemistry) use PBS, many changes (as much as 20) of a small volume of at least 10 times the volume of the brain or the piece.

Then dehydration in graded alcohols. Do it in steps 50%, 70%, 96%, 5 rinses each (again 5-10 volumes, 5-10 minutes per rinse under constant rocking). Then, this is critical, 100% ethanol, a freshly open bottle (absolute ethanol is extremely hygroscopic; it becomes hydrated very quickly after opening the bottle, especially in very humid climates). And then clearing in xylene or benzene or toluene, 5 more 5-10-min rinses. If everything runs properly the brain should become nearly transparent: if this does not happen something has gone wrong (see point 3 below). And then embedding in paraffin wax. Use low-temperature melting paraffin, and do at least x3 30-min rinses before doing the block. 

The critical points are:

1. Too much time in alcohol >70%, in clearing (xylene or benzene or toluene) or in hot paraffin means hardening the tissue a lot and this makes cutting very difficult. It is better to do many short baths (e.g. 10x5 minutes) rather than few long ones (e.g. 5x15).

2. Second, you should avoid bubbles near the tissue, this might also affect sectioning, and the tissue nearby the bubble can by damaged during sectioning. Might this have happen to you?.

3. Too less time in dehydration and clearing or the use of hydrated 100% alcohol might result in deficient embedding. If a portion of the tissue is not been properly dehydrated/cleared (it is not transparent after clearing) it has no paraffin inside and it breaks giving rise to a hole or a void area or a "crumbled" portion of tissue. Might this have happened to you?

Good luck

Fernando