[Histonet] Questions regarding tissue shrinkage during fixation and decalcification

[Phillips, Derrick]

Dear Histonet,



I am new to histonet, and relatively new to histology.  I have been digging through the histonet archives and doing searches on pubmed to try and answer some questions to a few problems I've been having.  It has been tough finding a definitive answer to my questions so I am writing my first email, hoping someone out there can help me out.  



I'm trying to section through the the head of an adult rat.  We've placed a recording electrode between the skull and the dura, and we are trying to section through all of the tissue to see how successful we are in electrode placement.  The first problem I have been having is the amount the brain is shrinking (at least I believe it is due to the brain shrinking).



I perfuse first with 0.9% saline, then fix with 10% formalin in PBS. 

Then it sits for 2 days in 10% formalin PBS + 20% sucrose. 

Afterward it is decalcified in 5% formic acid in DI for 4-5 days

then in 5% gelatin for 24 hours, followed by 10% gelatin, then placed in the fridge to harden.  After this it goes into -80, cut down, then sectioned in a cryostat. 



>From what I understand, fixing in formalin reduces the size of the brain tissue by about 20%.  I've read that during the perfusion, instead of a prewash in 0.9% saline, using 9.45% sucrose helps to maintain the extracellular space and reduce the amount of shrinking. (Brain extracellular space fixed for electron microscopy.  B. Cragg).  I am wondering if anyone else has used this protocol and been successful. 



There was a post in the histonet archives from a Matt McElwee asking about a decal procedure that doesn't shrink or alter the brain, but the reply I found said that it wasn't necessary for the experiment he was doing.  But it cited a protocol for decal by Gayle, but I was unable to locate this protocol.  Is it possible that I am getting additional shrinkage from the decal process in addition to my fixation?  If so, is there a way to reduce this? 



Another issue is the acceptance of the tissue to a glass slide.  The brain and muscle tissue seem to take to the glass slides well, but the problem I've been having is with the bone.  It tends to bow out and not adhere to the slide.  This can be quite frustrating.  I've tried charged slides, poly-l-lysine coated, silane coated, and superfrost plus slides, with minimal luck.  Is there anything that can be done to help keep the bone on the slides?



Finally, we are wanting to do triple fluorescent labelling of the tissue, the antigens need to remain intact.  We are looking for proteins, so if something were to happen to the RNA, or DNA I wouldn't be as concerned.  



The search for answers on these topics has been frustrating at best.  There seems to be a lot of variation in technique and I am unsure which will work the best for my needs.  Any advice would be greatly appreciated!!  Thank you!



Derrick Phillips

derric57 <@t> vetmed.wsu.edu