I need to fix my animals with 4% Para to do IHC on the sections later. But I also need to determine the integrity of the BBB and I have chosen the IV HRP method. I have read that most others use Karnovsky's Fixative (2% Paraformaldehyde, 2.5% Glutaraldehyde and 0.1M Buffer). Any suggestions? What are the benefits to using the Karnovsky's Fixative?
Daniel,
usually the "Karnovsky fixative" (concentrations you mention here = 'KARNOVSKY'S half strength fixative' cf: *) below ) is used if superior preservation of ultrastructure is to be achieved. If studying BBB and its integrity, perfusion fixation (IMHO) is highly recommended, buffered FA-GA-solution (i. e. , Karnovsky 1/2-strength or any other concentrations, or -better- Acrolein - FA-GA [Acrolein unfortunately seriously toxic and hazardous chemical] with the correct pH AND isotonic or slightly !hypertonic [by vehicle] and applied with the correct pressure to B-BRAIN-B-local area FOR combined LM, L-IHC,L-ICC as well as following ultrastructural examinations (e.g. TEM, SEM or AFM), if only for LM-morphology, L-IHC or L-ICC 2.5 - 4% (correctly buffered) PFA solution should be sufficient.
Possibly you should think about using Fixative in 0.13 M (PO4) buffer instead of 0.1M (search Google e. g. for < Karnovsky half strength > and or < Perfusion fixation brain [animal] benefit effect >
cf. also (just as one example out of 100s of publicatons):
*) http://www.ihcworld.com/_faq/histology-faq/fixation/f3.htm:
QUOTE:
"A neutral, buffered, isotonic formaldehyde-glutaraldehyde mixture should be fine for any kind of electron microscopy. Many workers like to use paraformaldehyde as the source of formaldehyde.
A classical mixture is M. J. Karnovsky's (J. Cell Biol. 27: 137A-138A, 1965). This is probably the most-cited unrefereed abstract! It contains approximately 4% formaldehyde and 5% glutaraldehyde in approximately 0.1 M phosphate or cacodylate buffer. Final pH = 7.2. If cacodylate (toxic!) is used, add calcium chloride (0.5 mg/ml) to improve preservation of membrane phospholipids.
Probably this fixative is frequently misquoted, and the literature is full of references to "half-strength Karnovsky," which probably means half the glutaraldehyde concentration. A glutaraldehyde concentration of 1 to 2% is commonly considered adequate in mixtures of this kind.
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
(kiernan[AT]uwo.ca)"
Best wishes and good luck (hopefully you'll get other more sophisticated replies from the experts)
Wolfgang
Daniel,
usually the "Karnovsky fixative" (concentrations you mention here = 'KARNOVSKY'S half strength fixative' cf: *) below ) is used if superior preservation of ultrastructure is to be achieved. If studying BBB and its integrity, perfusion fixation (IMHO) is highly recommended, buffered FA-GA-solution (i. e. , Karnovsky 1/2-strength or any other concentrations, or -better- Acrolein - FA-GA [Acrolein unfortunately seriously toxic and hazardous chemical] with the correct pH AND isotonic or slightly !hypertonic [by vehicle] and applied with the correct pressure to B-BRAIN-B-local area FOR combined LM, L-IHC,L-ICC as well as following ultrastructural examinations (e.g. TEM, SEM or AFM), if only for LM-morphology, L-IHC or L-ICC 2.5 - 4% (correctly buffered) PFA solution should be sufficient.
Possibly you should think about using Fixative in 0.13 M (PO4) buffer instead of 0.1M (search Google e. g. for < Karnovsky half strength > and or < Perfusion fixation brain [animal] benefit effect >
cf. also (just as one example out of 100s of publicatons):
*) http://www.ihcworld.com/_faq/histology-faq/fixation/f3.htm:
QUOTE:
"A neutral, buffered, isotonic formaldehyde-glutaraldehyde mixture should be fine for any kind of electron microscopy. Many workers like to use paraformaldehyde as the source of formaldehyde.
A classical mixture is M. J. Karnovsky's (J. Cell Biol. 27: 137A-138A, 1965). This is probably the most-cited unrefereed abstract! It contains approximately 4% formaldehyde and 5% glutaraldehyde in approximately 0.1 M phosphate or cacodylate buffer. Final pH = 7.2. If cacodylate (toxic!) is used, add calcium chloride (0.5 mg/ml) to improve preservation of membrane phospholipids.
Probably this fixative is frequently misquoted, and the literature is full of references to "half-strength Karnovsky," which probably means half the glutaraldehyde concentration. A glutaraldehyde concentration of 1 to 2% is commonly considered adequate in mixtures of this kind.
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
(kiernan[AT]uwo.ca)"
Best wishes and good luck (hopefully you'll get other more sophisticated replies from the experts)
Wolfgang
Short rule: fixation for electron microscopy needs glutaraldehyde, without or in cobination with paraformaldehyde (concentration of each substance determines quality for the type of tissue).
Light microscopy needs only formalin or paraformaldehyde. Colouration determines other type of fixatives, e.g. alcohol based. By the way Muss's answer is correct.
Best wishes and succes. Enrico Marani
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