Re: [Histonet] Fixed frozen non-paraffin mouse brain
Chuckle Thank you, John Kiernan Yes...I appreciate your filling in them gaps. I do understand/know themhaving read, over the years many of the Giants of Histology who gave me such inciteful/usable knowledge, including yourself. I just didn't want to bloat my "pennyworth" otherwise it would become several Poundsworth , chuckle. I am not worthy I recall having pp in unbuffered Formalin...we chucked it Well, in those days one emptied it down the sink! Sure, re Formic acid However, imho...that is a more theoretical problem, as Formalin was used up within a couple of weeks...in any of my Labs I used to check the pH once a week. In practice, there is much leeway in the use of Formalinprovided one is knowlegable regarding all the parameters? Sure, it is not the "best" fixing fluid butit all depends, as you intimated, on one's needs ( eg: Ultrastructural integrity v immunoreactivity) Thank you very much for your elucidations I always learn moream happy to I have the ORANGE book ( at work..I am on furlough) ...grrr...I fail to recall the most excellent author...on fixation. 1st edition I thoroughly enjoy/ed reading it It is another of my "Bibles"it expands my mind just like any Bible should...imho. A bible is like a learning curve: one must go back to it when in doubt but.go forward when in no doubt but, supported/stayed by that original knowledge Hence, I can see further by standing on the shoulders of the giants that came before me? Paraphrasingly...surely. Your opinion re the Orange-covered book ? Sure, there are many resources/publications regarding fixation...most are idiosyncraticimho. Respectfully Carl Carl Hobbs FIBMS Histology and Imaging Manager Wolfson CARD Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6813 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Fixed frozen non-paraffin mouse brain
And a very good pennyworth it is, Carl! You wrote, "... someone must've originally thought: 'Hang on, if we fix in commercially bought 40% Formalin, it's got 10% methanol added (to slow rate of formaldehyde repolymerisation) ...that will compete with formaldehyde fixation. So, we get coagulative and additive fixation. That is not good, folkslet's get pure and use depolymerised paraformaldehyde: pure methylene glycol polymer'". That's almost how it came about: let's get pure. A fixative made from PFA should have the same composition every time it is freshly prepared. The 10% of methanol (MeOH) in formalin (40% HCHO) isn't enough to coagulate proteins, and neither is the 1% MeOH in 10% formalin (with 4% HCHO). You need 60-70% alcohol to coagulate proteins, viruses etc. Formalin also contains some formic acid; the amount increases with age, from oxidation of the aldehyde by air. Dilution with water always gives an acidic solution. Marble chips can be added bring the pH up to neutrality. Buffering also takes care of the formic acid and can provide a neutral (pH7) or a "physiological" (pH7.4) fixative solution. The usual phosphate buffer also makes the fixative solution approximately iso-osmotic with mammalian extracellular fluid. Before the 1960s, dilution of formalin with with saline (0.9% NaCl) provided "formal saline", which had some advantages over 4% aqueous formaldehyde. See books by J. R. Baker, which are available as free downloads from http://archive.com. Polymerization also increases with age. That's why you see a white precipitate in bottles of formalin stored for a long time. The precipitate is paraformaldehyde (PFA); its presence reduces the amount of formaldehyde that can be easily released by simple dilution of the formalin with water. According to R. Cares (1945: A note on stored formaldehyde and its easy reconditioning. J. Tech. Methods & Bull. Int. Ass. Med. Museums 25, 67-70), milky formalin can be cleared by autoclaving, for 30 m in Kilner jars. I wonder if anyone else has done this? John Kiernan Anatomy & Cell Biology UWO, London, Canada = = = From: Hobbs, Carl via Histonet Sent: 05 July 2020 14:25 To: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Fixed frozen non-paraffin mouse brain Prof. Kiernan, as usual, provides us all with such a depth/breadth of particular information/advice. His Histological and Histochemical methods BIBLE is still my favourite read. Respect Most researchers fix in depolymerised Paraformaldehyde because someone must've originally thought: " Hang on, if we fix in commercially bought 40% Formalin, it's got 10% methanol added ( to slow rate of formaldehyde repolymerisation) ...that will compete with Formaldehyde fixation. So, we get coagulative and additive fixation. That is not good, folkslet's get pure and use depolymerised Paraformaldehyde: pure methylene glycol polymer" I am sure Professor Kiernan can correct my inaccuracies! Anyway..I've never noticed any difference: I've worked in diagnostic labs ( unfixed frozen muscle/renal/rectal bx) and also research labs ( unfixed/ fixed frozen tissues) using both fixing solutions I have not noticed any IHC/IF difference in reactivity. Many primary abs do NOT work even with fixed/unfixed frozensome of them WILL need HIER ( at 90C rather than M/W or pressure cooker AR but, only fixed frozen of course), imho. Part of the problem is whether the antigen is linear or 3D...sorry for simplicity. I can successfully snap-freeze fixed/unfixed rat/ms brain hemispheres without using sucrose ( success measured by lack of holes at the LM level). This is because I was trained in a diagnostic lab to freeze fast but, effectively. It is a technique that requires experience for consistency of successsometimes I fail! The reason most use 20/30% sucrose is to give poor a snap-freezing technique a chance to avoid ice-crystal artefact, as stated by Kiernan). Sucrose is no panacea.technique is everything. My pennyworth-illy Carl Carl Hobbs FIBMS Histology and Imaging Manager Wolfson CARD Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6813 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Fixed frozen non-paraffin mouse brain
Prof. Kiernan, as usual, provides us all with such a depth/breadth of particular information/advice. His Histological and Histochemical methods BIBLE is still my favourite read. Respect Most researchers fix in depolymerised Paraformaldehyde because someone must've originally thought: " Hang on, if we fix in commercially bought 40% Formalin, it's got 10% methanol added ( to slow rate of formaldehyde repolymerisation) ...that will compete with Formaldehyde fixation. So, we get coagulative and additive fixation. That is not good, folkslet's get pure and use depolymerised Paraformaldehyde: pure methylene glycol polymer" I am sure Professor Kiernan can correct my inaccuracies! Anyway..I've never noticed any difference: I've worked in diagnostic labs ( unfixed frozen muscle/renal/rectal bx) and also research labs ( unfixed/ fixed frozen tissues) using both fixing solutions I have not noticed any IHC/IF difference in reactivity. Many primary abs do NOT work even with fixed/unfixed frozensome of them WILL need HIER ( at 90C rather than M/W or pressure cooker AR but, only fixed frozen of course), imho. Part of the problem is whether the antigen is linear or 3D...sorry for simplicity. I can successfully snap-freeze fixed/unfixed rat/ms brain hemispheres without using sucrose ( success measured by lack of holes at the LM level). This is because I was trained in a diagnostic lab to freeze fast but, effectively. It is a technique that requires experience for consistency of successsometimes I fail! The reason most use 20/30% sucrose is to give poor a snap-freezing technique a chance to avoid ice-crystal artefact, as stated by Kiernan). Sucrose is no panacea.technique is everything. My pennyworth-illy Carl Carl Hobbs FIBMS Histology and Imaging Manager Wolfson CARD Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6813 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Fixed frozen non-paraffin mouse brain
Dear Ed, Adequate fixation is important. Formaldehyde penetrates quickly but reacts slowly with proteins. 4% formaldehyde made by depolymerizing paraformaldehyde (an insoluble high polymer) is the same as formaldehyde made by 10X dilution of formalin (a mixture of soluble low polymers). For cryoprotection the sucrose must thoroughly penetrate the fixed specimen, which must sink in the concentrated solution. Your Swiss cheese artifact is due to slow freezing. In badly frozen brains the ice crystal holes are as big as large neurons and the tissue architecture is destroyed. Unless specimens are really tiny and frozen super-fast (special methods for electron microscopy), ice crystals always form and show later as holes in the sections. You get bigger holes with bigger specimens and slower freezing. With very fast freezing, a skilled technician can get good cryostat sections even of small unfixed specimens such as muscle biopsies. These are needed for enzyme activity histochemistry methods used in diagnostic pathology and in research. Cryoprotection of fixed specimens slows the growth of ice crystals. With luck, the holes are too small to interfere with light microscope studies of sections. In neuroscience research, quite thick frozen sections of samll animals' brains have been the norm for more than 50 years. About 20 years ago I wrote a chapter that gave some quite detailed instructions and explanations, with references. (Don't do anything important just because a chapter or a review says so; check at least some of the refs!) Here is the reference. Kiernan, J. A. 2002. Freezing and fixation. Chapter 8 in Microscopy and Histology for Molecular Biologists. A User's Guide, ed. Kiernan, J. A. & Mason, I. G. pp. 103-143. London: Portland Press. ISBN 1855781417. Your university's library in Urbana might have the book. It's out-of-print with its publisher. There are used copies on the web for much less than the original price. John Kiernan Emeritus neuroanatomist and histochemist London, Canada https://www.schulich.uwo.ca/anatomy/people/bios/emeriti/kiernan_john.html = = = From: Roy, Edward J via Histonet Sent: 04 July 2020 20:08 To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Fixed frozen non-paraffin mouse brain As a research lab, we sometimes would like to use paraformaldehyde-fixed but non-paraffin embedded tissues; paraffin embedding alters antigens and necessitates antigen retrieval, but simple fixation does not. We have done the traditional 30% sucrose before OCT and freezing, with cryostat sectioning, but results are inconsistent, sometimes producing Swiss-cheese brains. Does anybody have an alternative to 30% sucrose that is more reliable? I didn’t see anything in the Archives after a search for “30% sucrose”. Thanks very much, Ed Roy Edward J. Roy, PhD Professor Emeritus Department of Molecular and Integrative Physiology University of Illinois at Urbana-Champaign Urbana, IL 61801 217 333-3375 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Fixed frozen non-paraffin mouse brain
Hi Ed, I have been using the 30% sucrose technique to cryoprotect animal tissue for over 40 years without any problem. Did the tissue sink to the bottom of the specimens jar? After sinking, I blot the excess sucrose from the tissue on a paper towel before transfering to OCT. What is your procedure to freeze the tissue? Thank you, Tina Van Meter Scripps Research Histology Core Manager Jupiter, FL On Sat, Jul 4, 2020, 9:26 PM Roy, Edward J via Histonet < histonet@lists.utsouthwestern.edu> wrote: > As a research lab, we sometimes would like to use paraformaldehyde-fixed > but non-paraffin embedded tissues; paraffin embedding alters antigens and > necessitates antigen retrieval, but simple fixation does not. We have done > the traditional 30% sucrose before OCT and freezing, with cryostat > sectioning, but results are inconsistent, sometimes producing Swiss-cheese > brains. Does anybody have an alternative to 30% sucrose that is more > reliable? I didn’t see anything in the Archives after a search for “30% > sucrose”. > Thanks very much, > Ed Roy > > > Edward J. Roy, PhD > Professor Emeritus > Department of Molecular and Integrative Physiology > University of Illinois at Urbana-Champaign > Urbana, IL 61801 > 217 333-3375 > > > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Fixed frozen non-paraffin mouse brain
You need to add sucrose to your PFA we use 4%PFA+4%Sucrose to fix and then cryoprotect in 30% sucrose which all need to be prepared in Phosphate buffer solution NO saline if you message me directly I would be happy to share our SOP this was a very hard thing to learn not a lot in technical references about this process. Sent from my iPhone > On Jul 4, 2020, at 9:09 PM, Roy, Edward J via Histonet > wrote: > > As a research lab, we sometimes would like to use paraformaldehyde-fixed but > non-paraffin embedded tissues; paraffin embedding alters antigens and > necessitates antigen retrieval, but simple fixation does not. We have done > the traditional 30% sucrose before OCT and freezing, with cryostat > sectioning, but results are inconsistent, sometimes producing Swiss-cheese > brains. Does anybody have an alternative to 30% sucrose that is more > reliable? I didn’t see anything in the Archives after a search for “30% > sucrose”. > Thanks very much, > Ed Roy > > > Edward J. Roy, PhD > Professor Emeritus > Department of Molecular and Integrative Physiology > University of Illinois at Urbana-Champaign > Urbana, IL 61801 > 217 333-3375 > > > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.com%2Fv3%2F__http%3A%2F%2Flists.utsouthwestern.edu%2Fmailman%2Flistinfo%2Fhistonet__%3B!!HXCxUKc!jkI5NdLT3gGvmm9DK87HJOVfSDogjkNNjK6aobOZKeIdVbQSf5Y3zN9OCgerig%24data=01%7C01%7Cportera%40msu.edu%7C7cacd5e670c74a9bcac208d8208004ac%7C22177130642f41d9921174237ad5687d%7C0sdata=CE67aooP%2BsILBNDemkXRjDcNpQv4XmQa7eb%2BBP4d8HA%3Dreserved=0 > ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Fixed frozen non-paraffin mouse brain
As a research lab, we sometimes would like to use paraformaldehyde-fixed but non-paraffin embedded tissues; paraffin embedding alters antigens and necessitates antigen retrieval, but simple fixation does not. We have done the traditional 30% sucrose before OCT and freezing, with cryostat sectioning, but results are inconsistent, sometimes producing Swiss-cheese brains. Does anybody have an alternative to 30% sucrose that is more reliable? I didn’t see anything in the Archives after a search for “30% sucrose”. Thanks very much, Ed Roy Edward J. Roy, PhD Professor Emeritus Department of Molecular and Integrative Physiology University of Illinois at Urbana-Champaign Urbana, IL 61801 217 333-3375 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
