[Histonet] Re: can you defrost and fix muscle tissue?

[Hobbs, Carl]

Your project reads ill-thought out!

I agree with Elizabeth re defrosting.
Should be fine.

Yes, if your freezing is slow, you will most likely have ice-crystal artefact.​
You will see this in the Pwax sections.
You have large pieces of muscle.
Good that you fixed for longer.
Imho, if you are not requiring Immunostaining, the longer the fixation...the better ( within reason)

I would assume your problems are a result of UNDER processing.
Muscle pieces that size, I would always process to Pwax over 40hr schedule.
What was your processing schedule?

I often Pwax process 1x1x0.5cm pig skeletal muscle and have no problems cutting....nope, no ice involved.
Just a sharp knife and, a gentle rub of the block face every section, using 20% alcohol -damped soft tissue.

If you are examining blood vessels....you HAVE to perfuse-fix, imho.....for best results.
When you get damn fine sections:
Do an H&E and an HVG, initially.
As Elizabeth stated....stick to Formalin fixation.

NB: PFA is not a fixative.
It is a polymer of methylene glycol and is...a powder!
When dissolved it forms multiple forms : methylene glycol is the monomer.
What you buy when you get "40% Formalin" is methylene glycol in solution ( Formaldehyde gas dissolves in water to ~ 40% saturation).
Dilute that 1/10 and you get 10% Formalin ( same as 4% "PFA")
The ONLY reason them Molecular biologists/chemists decided to use PFA is cos they have NO idea re Formalin/methylene glycol fixation!
Neither do I, chuckle.
Well.....the reason they decided to use Paraformaldehyde powder is because the immediate product is , more or less, pure methylene glycol.
In bought 40% Formalin, they add a small amount of alcohol to retard formation of Formic acid and polymerised methylene glycol( PFA).
So.....them geezers thought: OMG...alcohol! No....it will change the chemistry of fixation!
Sure....them geezers NEVER bothered to look at most clinical labs in the world who buy/use ~40% Formalin to make up their 10% Formalin....with great success!!
Imho, nobody has produced a convincing raison d'être for adding PBS.
It is added because we think of physiology....add PBS to a fix and we get ..."better" fixation.
Chuckle....Formalin ( methylene glycol) KILLS cells.
Adding PBS...hmmm.
I still do.....chuckle.

Curiously, as ever.

carl


 
 
Carl Hobbs FIBMS 
Histology and Imaging Manager 
Wolfson CARD 
Guys Campus, London Bridge  
Kings College London 
London 
SE1 1UL 
  
020 7848 6813