Carnoy is a water-free fixative. So dehydration should take place de facto during fixation. The absolute ethanol afterwards replaces the fixative and completes dehydration. So the duration depends on the time, that is needed for full infiltration of the tissue. And infiltration-time depends on the size of your tissue. (Maybe you have to trim your specimens into smaller pieces?)

For FFPE processing the standard protocols often have two steps with one hour in absolute ethanol each for tissue with diameter up to 5 mm. The xylen step is the same. Using two baths is always recommended.

Shrinking can be caused by Carnoy itself (because it is water-free), prolonged absolute ethanol, xylene (solves fat) and too hot paraffin.

So as always in histotechnic - it depends. - on size, tissue-composition, ...

good luck

Gudrun

Carnoy is a water-free fixative. So dehydration should take place de facto during fixation. The absolute ethanol afterwards replaces the fixative and completes dehydration. So the duration depends on the time, that is needed for full infiltration of the tissue. And infiltration-time depends on the size of your tissue. (Maybe you have to trim your specimens into smaller pieces?)

For FFPE processing the standard protocols often have two steps with one hour in absolute ethanol each for tissue with diameter up to 5 mm. The xylen step is the same. Using two baths is always recommended.

Shrinking can be caused by Carnoy itself (because it is water-free), prolonged absolute ethanol, xylene (solves fat) and too hot paraffin.

So as always in histotechnic - it depends. - on size, tissue-composition, ...

good luck

Gudrun

Hi - I agree with the above, but if you are going to store the tissue for any time before processing, then remove it from the Carnoy's fixative and store in 70% alchol. The tissue will degrade fairly quickly if stored in abosolute alcohol or Carnoy's for extended periods.

Otherwise, process immediately after fixation from absolute alcohol.

Best wishes Hedley

Carnoy's fixative in an alcohol-based fixative (non-formalin).  The chloroform and acetic acid helps the integrity of the tissue cells from shrinking compared to alcohol-fixed tissues.  The chloroform also soften the tissues and keeps it from becoming brittle.  It's an excellent fixative for DNA/RNA.   After processing and embedding tissues in paraffin , tissue sections usually does not need a heat-retrieval method (antigen retrieval) as does most formalin-based fixatives.   However, certain antigens may not work as well in Carnoy's as formalin-fixed tissues.

Thank you very much for your help everybody!

Hi,

I am also using carnoy fixative, but during the steps of tissue processing, the tissue is getting detached from the blotting paper. subsequently the orientation of the tissue is no more possible. what adhering surface do you use for keeping the tissue oriented even after processing?

We don't use adhering paper or similar during processing (FFPET). For orientation we use tissue marking ink or lay the tissue between tissue-sponges.

Usually the tissue itself tells the orientation because the "smooth" and more "shiny" side, where the tissue was cut, is the surface of interest and has to be down in the embedding mold.

Thank you for your reply. My sample is a human biopsy specimen. Because of its very small size, I think that using ink will stain my entire tissue. Correct me, if I am wrong!

I have thought of using tissue sponges. I want to collect each biopsy specimen individually. I am sharing some links with you, tell me whether they will serve the purpose or you can suggest me others.

https://www.thomassci.com/Equipment/Histology/_/HISTOLOGYCYTOLOGY-ACCESSORIES?q=Tissue%20Cassettes

https://www.medline.com/product/Microsette-I-Biopsy-Cassettes-by-Simport-Scientific/Biopsy-Cassettes/Z05-PF34095?question=&index=P9&indexCount=9

Also, once the tissue enters fixative, it turns white, there is no way to make any assumption of the smooth and its shiny surface, in my case.

Previously, during sample collection,