The uneven fixation may be due to the volume of the piece that was fixed. 4% formaldehyde (or paraformaldehyde) penetrates slowly in the tissues to be fixed. So, the more central areas begin to suffer autolysis. My advice is to cut a testis through a sagittal plane and then make serial (transversal) sections of each half. Each serial section should be 4 mm-thick, 5 mm in the maximum. If necessary, each serial section could be segmented following cutting planes perpendicular to the transversal sections.

You could use another fixative, Bouin's fluid, but the thickness of the material to be fixed is of utmost importance (4 mm-thick).

I wish you good luck with your research.

Dear Jose, thank you so much for your suggestion, it's very helpful.  To avoid the separation of testicular tissue during cutting whether tunica albuginea need is perforated using a needle 5 hours before cutting to small size?   With the procedure, the fixative fluid more quickly penetrate the testicular tissue through those several holes and testicular tissue will faster hardened (fixed).

Dear Sri, yes, you could perforate the tunica albuginea with a needle, but the holes should be at approximately equal distance between them to allow an uniform fixative penetration. Also, the puncture should perforate only the tunica albuginea to avoid damaging testicular parenchyma. After 4-5 hours, the parenchyma should be hardened enough to perform the longitudinal section (first) and the serial sections (after).

Dear Sri,

Use of Davidson's fluid will yield superior morphology but you will still suffer from shrinkage. Small holes are a good idea too. What would you like to analyze in testis?

Best,

Ivan

Dear Sri,

My idea is that you can in situ perfuse the whole body with 10% formalin after having bled through right common carotid artery. Procedure is that when bleeding is completed, you can introduce 10% formalin through the same route. After doing this for 30 min, the whole body including testis will be fixed, then you can remove testes. Although, other tissues will be spoiled. 

I suggest Bouins  fluid for the testis fixation, though you would have a yellow tint to the section. Otherwise you may need to perfuse the animal with the paraformaldehyde.

But most importantly the size of the tissue is very paramount to which of the methods you may wish to apply

Dear Jose, Ivan, Muhammad, and Moses. Thank you very much for your explanation and suggestion. I will use testicular organ to detected several active compounds that play a role in spermatogenesis immunohistochemically.

Associated with Bouin's and Davidson's fluid, whether they do not damage the active compounds to be detected? Some literature stated that Bouin's fluid more rapid tissue penetration and more appropriate for staining tissue structure. For Davidson's fluid, I have not tried it yet as fixatives, however it needs to be tried.

Yes, perfusion methods is better for tissue fixation but I usually do that method for small animals whereas for large animals I prefer immersion fixation for testicular tissue obtained from slaughterhouses.

Thank you.

Best regards

Dear Sri:

To the best of my knowledge, for immunocytochemistry, one of the best (or more used) fixatives is 10% buffered neutral formalin (i.e., 4% buffered neutral paraformaldehyde) in immersion. Depending of the molecules you want to detect, sometimes frozen sections are mandatory,

Dear Jose

Thank you for valuable information. One more question, how long should the time for immersed testicular tissue in 10% buffer neutral formalin?

Best regards,

Dear Sri:

The best time is 24 hours. Less time causes underfixation with consequent autolysis. You can fix the tissues until 48 hours, but longer fixation times render epitope retrieval more difficult and imperfect, with uneven immunocytochemical staining.

Dear Sri,

In Bouin's fluid=24 hrs

In 10% buffer neutral formalin= 7 days

Zamboni's Solution= 7 days

I think so.

Dear Muhammad,

Thank you so much for your information.

Best regards

Dear Jose,

Thank you so much for your explanation. It is valuable for support my works.

Best regards

Hi Sri,you may fix it in modified Davidson's fluid for 24 hrs and give brief rinse under tap water and put it in 10%NBF. Don't forget to puncture the testis with a pin before putting it into fixative to allow optimum penetration of fixative.hope this will help

Dear Nataraju, thank you very much for the advice. Is the fixation time in 10%NBF also 24 hours?

Best regards

At least keep the tissues in 10%NBF for 72 hrs then you can do further processing.

Thank you so much for the fixation procedure.

Best regards.

Bouin solution for 24 hours is best