Re: [Histonet] Help! NBF fixed tissue and subsequent first step for dehydration.

From:Rene J Buesa

  Although there are numerous considerations and theories regarding the adverse effects that osmotic and liquid flow currents have on the cellular morphology it is a fact that the water in the cells has to be eliminated and that the space between all its constituents have to be replaced by paraffin. To get to that point, as you well know, the water has to be extracted generally by alcohol, the alcohol has to be exchanged by an antemedium miscible with both ethanol and paraffin and at the end the paraffin has to infiltrate the tissue.
  There are several "rules of the thumb" (ROT) for the whole process, that will vary with the type of tissue, being some more "resistant" to those changes (like connective tissue) than others (glands, like the pancreas in your case, and whole embryos).
  The first ROT is that the "gentler" the exchange, the better meaning that the dehydratoin should start with the lowest concentration possible or allowed by your tissue processor and the number of station it has. Many years ago (about 50) I used to work with lizard embryos and I designed a small bottle with a cork with 2 perforations. Through the perforations I inserted 2 glass tubes, the "IN" touched the bottom, and the "OUT" was almost at the surface. After washing the embryos I placed them in the bottle and let alcohol "IN" dropping until after 24-48 hours all the water had been substituted by 100% EthOL and the embryos were dehydrated very gently.
  The 2nd ROT is that when going from one reagent to the next use one step that contains half and half, if going from 100%EthOL to xylene, 1 step should be 1:2 of each reagent. In the same way at the end of my career I stopped using xylene and changed to mineral oil with the intermediate steps of 1:2 of consecutive reagents.
  Now about your question on the "cracking" appearence (I gather that it looks as if the cells look like broken crackers with "breaking" lines through the cytoplasm) this can be due either to excessive dehydration or could be due to the antemedium (the one between the alcohol and the paraffin).
  Delicate tissues require delicate procedures and the best approach is to get several samples and process them with different protocols to find out the best.
  Also it is not a good practice for you to receive semiprocessed tissues, you will never know how they did it. Try to get the tissues in the fixative and then you will for certain know how you processed it.
  Escuse me this long answer, I only hope that it will help you. Maybe the head of the research is right but you will never know until you control the whole process.
  Rene J.

Lorraine Rolston  wrote:
  I would appreciate any advice concerning possible detrimental effects that can be caused to the morphology of NBF fixed tissue when placed directly into 70%ethanol from NBF. This tissue (pancreas, spleen, kidney)is for research purposes (not clinical diagnosis) and good preservation of cell morphology is of extreme importance.
One of the research groups that uses our processing facility brings the tissues to us immersed in 70% ethanol ready for processing.They are then embedded and cut for H&E staining.
Before their tissues are bought to the lab at the 70% ethanol step, they are washed in water after fixation, then immersed in 50%alcohol. At some stage this protocol has been changed and the 50%ethanol step has been omitted. Coinciding with this step being discontinued has come the appearance of bad cracking of the tissue.The head of the research group now feels that the changes to the morphology of these samples is caused by the jump from the NBF fixative to 70%ethanol.
The pancreas samples are processed separately using chloroform for clearing.
I have yet to view the slides but may be able to submit a picture(if given permission) when I do.
Any helpful advice or ideas would be very much appreciated.
Thanks in advance, Lorraine.

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