Re: [Histonet] ammonium bromide in fixative
Thank you for such a detailed response. It is doubtful I would have found this information without you. The National Histology Society online learning center is one of my study resources, this is where the question came from. They also include several mercuric fixatives that we would never use in our lab in this day and age. Perhaps the material has not been updated for a number of years. I have not yet heard from any recent exam takers about the content of old techniques. I find them interesting, but don't know how much time to devote to them. Katherine S Walters Histology Director Central Microscopy Research Facility University of Iowa 76 Eckstein Medical Research Building 431 Newton Road Iowa City, Iowa 52242 319-335-8142 Facility Website: http://cmrf.research.uiowa.edu/ From: John Kiernan [mailto:jkier...@uwo.ca] Sent: Friday, June 12, 2015 11:58 PM To: Walters, Katherine S; Histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] ammonium bromide in fixative Formalin with ammonium bromide is the fixative prescribed for traditional silver and gold techniques for staining neuroglial/x cells in free-floating frozen sections. Adding ammonium bromide lowers the pH of the fixative because of a chemical reaction with formaldehyde. An Argentinian histologist/x called Lascano showed in the 1940s that any sufficiently acidified formaldehyde solution (pH 1.5) was just as good as FAB for Cajal's gold-sublimate (astrocytes) and for silver carbonate methods (oligodendrocytes, microglia). The reaction that lowers the pH is 4NH4+ + 6HCHO --- C6H12N4 + 6H2O + 4H+ (Subscripts and superscripts in this equation will not be honoured in this email!) The 4H+ lowers the pH. The other product, C6H12N4 is hexamethylenetetramine, also known as hexamine in Britain and methenamine in the USA, and used in various histochemical staining methods, notably Grocott's method for fungal cell-walls in sections of animal (including human) tissues. The bromide (Br-) ion of NH4Br is irrelevant, in the FAB fixative and also in Globus's pretreatments (dilute ammonia followed by dilute hydrobromic acid). Globus's bromuration treatment was applied to frozen sections of pieces of CNS that had been fixed in non-acidified formalin. The references for Lascano's work are: Lascano, E.F. (1946a). Influencia del pH en la impregnacion argentica del tejido nervioso. Archivos de la Sociedad Argentina de Anatomia Normal y Patologica 8:105-114. Lascano, E.F. (1946b). Importancia del pH en la fijacion del tejido nervioso. Creacion artificial de fijadores/x tipo formol-bromuro y formol-nitrato de urano de Cajal. Archivos de la Sociedad Argentina de Anatomia Normal y Patologica 8:185-194. Lascano, E.F. (1946c). Influencia del pH del fijador en la coloracion argentica del tejido nervioso. Archivos de la Sociedad Argentina de Anatomia Normal y Patologica 8:272-276. Not everyone agreed with Lascano! Polak, M. (1948). Sobre la importancia del bromuro de amonio de la solucion fijadora de Cajal en la impregnacion argentica del tejido nervioso. Archivos de la Sociedad Argentina de Anatomia Normal y Patologica 10:224-234. Do you really need this information to pass your HTL qualifying exam? It isn't easily found in books or with Google Scholar. I came across these papers quite by chance when looking over some old journals that UWO's library had set aside for throwing out (Gasp!), about 1980. Yes, they had hit a new low! Their services have, however, been exceptionally good for the last 10-15 years. Does anyone still use either Cajal's method for astrocytes or traditional silver carbonate methods to stain oligodendrocytes and microglia? They are difficult for several reasons, take up time, and require an old-fashioned freezing microtome to cut and collect the rather thick sections that are needed. Reliable antibodies for immunostaining glial cell-types have been available for many years, and they work on any kind of section. You need some thickness to appreciate the 3D shapes of astrocytes and oligodendrocytes, however they are stained. There's plenty of histo-history in the traditional neuroglia stains, which defined the cell-types for identification by electron microscopy and immunohistochemistry. John Kiernan Anatomy Cell Biology University of Western Ontario London, Canada = = = On 11/06/15, Walters, Katherine S katherine-walt...@uiowa.edumailto:katherine-walt...@uiowa.edu wrote: Hi all, I am studying to take the HTL certification test and ran across a reference to Formalin Ammonium Bromide. I see that it is very good for central nervous tissue fixation, it must be made fresh and that its pH is 1.5. Does anyone happen to know the reason for ammonium bromide in this fixative? I have been looking online and this has not been explained. Also, has anyone taken this test lately? I am curious as to how much old techniques, such as mercuric fixatives will be included
Re: [Histonet] ammonium bromide in fixative
Formalin with ammonium bromide is the fixative prescribed for traditional silver and gold techniques for staining neuroglial/x cells in free-floating frozen sections. Adding ammonium bromide lowers the pH of the fixative because of a chemical reaction with formaldehyde. An Argentinian histologist/x called Lascano showed in the 1940s that any sufficiently acidified formaldehyde solution (pH 1.5) was just as good as FAB for Cajal's gold-sublimate (astrocytes) and for silver carbonate methods (oligodendrocytes, microglia). The reaction that lowers the pH is 4NH4+ + 6HCHO --- C6H12N4 + 6H2O + 4H+ (Subscripts and superscripts in this equation will not be honoured in this email!) The 4H+ lowers the pH. The other product, C6H12N4 is hexamethylenetetramine, also known as hexamine in Britain and methenamine in the USA, and used in various histochemical staining methods, notably Grocott's method for fungal cell-walls in sections of animal (including human) tissues. The bromide (Br-) ion of NH4Br is irrelevant, in the FAB fixative and also in Globus's pretreatments (dilute ammonia followed by dilute hydrobromic acid). Globus's bromuration treatment was applied to frozen sections of pieces of CNS that had been fixed in non-acidified formalin. The references for Lascano's work are: Lascano, E.F. (1946a). Influencia del pH en la impregnacion argentica del tejido nervioso. Archivos de la Sociedad Argentina de Anatomia Normal y Patologica 8:105-114. Lascano, E.F. (1946b). Importancia del pH en la fijacion del tejido nervioso. Creacion artificial de fijadores/x tipo formol-bromuro y formol-nitrato de urano de Cajal. Archivos de la Sociedad Argentina de Anatomia Normal y Patologica 8:185-194. Lascano, E.F. (1946c). Influencia del pH del fijador en la coloracion argentica del tejido nervioso. Archivos de la Sociedad Argentina de Anatomia Normal y Patologica 8:272-276. Not everyone agreed with Lascano! Polak, M. (1948). Sobre la importancia del bromuro de amonio de la solucion fijadora de Cajal en la impregnacion argentica del tejido nervioso. Archivos de la Sociedad Argentina de Anatomia Normal y Patologica 10:224-234. Do you really need this information to pass your HTL qualifying exam? It isn't easily found in books or with Google Scholar. I came across these papers quite by chance when looking over some old journals that UWO's library had set aside for throwing out (Gasp!), about 1980. Yes, they had hit a new low! Their services have, however, been exceptionally good for the last 10-15 years. Does anyone still use either Cajal's method for astrocytes or traditional silver carbonate methods to stain oligodendrocytes and microglia? They are difficult for several reasons, take up time, and require an old-fashioned freezing microtome to cut and collect the rather thick sections that are needed. Reliable antibodies for immunostaining glial cell-types have been available for many years, and they work on any kind of section. You need some thickness to appreciate the 3D shapes of astrocytes and oligodendrocytes, however they are stained. There's plenty of histo-history in the traditional neuroglia stains, which defined the cell-types for identification by electron microscopy and immunohistochemistry. John Kiernan Anatomy Cell Biology University of Western Ontario London, Canada = = = On 11/06/15, Walters, Katherine S katherine-walt...@uiowa.edu wrote: Hi all, I am studying to take the HTL certification test and ran across a reference to Formalin Ammonium Bromide. I see that it is very good for central nervous tissue fixation, it must be made fresh and that its pH is 1.5. Does anyone happen to know the reason for ammonium bromide in this fixative? I have been looking online and this has not been explained. Also, has anyone taken this test lately? I am curious as to how much old techniques, such as mercuric fixatives will be included? Thank you, Katherine S Walters Histology Director Central Microscopy Research Facility University of Iowa 76 Eckstein Medical Research Building 431 Newton Road Iowa City, Iowa 52242 319-335-8142 Facility Website: http://cmrf.research.uiowa.edu/ Notice: This UI Health Care e-mail (including attachments) is covered by the Electronic Communications Privacy Act, 18 U.S.C. 2510-2521, is confidential and may be legally privileged. If you are not the intended recipient, you are hereby notified that any retention, dissemination, distribution, or copying of this communication is strictly prohibited. Please reply to the sender that you have received the message in error, then delete it. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list
[Histonet] ammonium bromide in fixative
Hi all, I am studying to take the HTL certification test and ran across a reference to Formalin Ammonium Bromide. I see that it is very good for central nervous tissue fixation, it must be made fresh and that its pH is 1.5. Does anyone happen to know the reason for ammonium bromide in this fixative? I have been looking online and this has not been explained. Also, has anyone taken this test lately? I am curious as to how much old techniques, such as mercuric fixatives will be included? Thank you, Katherine S Walters Histology Director Central Microscopy Research Facility University of Iowa 76 Eckstein Medical Research Building 431 Newton Road Iowa City, Iowa 52242 319-335-8142 Facility Website: http://cmrf.research.uiowa.edu/ Notice: This UI Health Care e-mail (including attachments) is covered by the Electronic Communications Privacy Act, 18 U.S.C. 2510-2521, is confidential and may be legally privileged. If you are not the intended recipient, you are hereby notified that any retention, dissemination, distribution, or copying of this communication is strictly prohibited. Please reply to the sender that you have received the message in error, then delete it. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
