Re: [Histonet] Problems with mouse brains
Hi Julie: You mentioned that you are buying your fixative from a commercial supplier but that My first thought is that there was difference in formalin... but that is not the case. How do you know? You did not make the fixative solution so you really have no idea. Not to berate Fisher Scientific, I have been a loyal customer since grad school. Maybe someone had a bad day or was interrupted during the mixing. Mixing buffered formalin in you lab is so easy and you retain control of the most important step in histology. Geoff Randolph-Habecker, Julie wrote: Folks, I need some input on a problem we're seeing with some mouse brain samples. The samples are from new born mice P0 to P14 which have been fixed in 10% NBF (Fisher brand and well within expiration date) for 72 hours. They are then transferred to 70% etoh and processed on an 8 hour process. After the tissue is embedded, we are taking sagital sections, drying them overnight, and then baking overnight at 60C. They are then stained with HE. This has been working great but now all of a sudden we have very light HE staining, nuclear bubbling, and extensive tissue cracking. I also have noticed some formalin pigment. My first thought is that there was difference in formalin or time in formalin but that is not the case. I also wondered if the tissue might be exposed to excessive heat. We checked all of the temperatures in our processor, embedding center, and water baths - all were within tolerance. Any ideas what might cause all three artifacts? Could it be from inadequate paraffin infiltration? Any help would be greatly appreciated! Thanks, Julie Julie Randolph-Habecker, Ph.D. Staff Scientist - Director Experimental Histopathology Shared Resource Fred Hutchinson Cancer Research Center 1100 Fairview Ave, N. DE-360 (Please note new location) Seattle WA 98109-1024 206-667-6119 jhabe...@fhcrc.org ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- -- ** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583 mcaul...@umdnj.edu ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Problems with mouse brains
Hello Kemlo, I am Portuguese-Spanish so we use the word tampón that I think I miss translated to tamponated..really do not know if the word exists! but apparently it does not! :) I want to mean when you add salt to the formol dilution in destilated water in order to prepare your 10% buffered formalin... a believe the word is buffered So sorry..for my wrong translationI was in a hurry...what I want to say is THAT YOUR FORMALIN IN NOT WELL BUFFERED :) thank you! and Yes, you should be carefull your brain is well fixated in formalin at least for 24 hour. 2009/4/5 kemlo ke...@f2s.com Agree, poor formalin fixation will allow nuclear bubbling which some authorities put down to the effects of heat on the sub optimally stabilised proteins. What a marvellous phrase your formol is not well tamponated. Wish I had thought of that.. I will of course steal it if you allow such plagiarism? -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Ana Resendes Sent: 04 April 2009 15:16 To: Randolph-Habecker, Julie Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Problems with mouse brains have very light HE staining: first you should check if either your eosin or haematoxilin are over used nuclear bubbling: can be a lot of things but can be inclusion. Brain sections are big so make at least a 24 h inclusion process. and extensive tissue cracking: could be over heating or bad inclusion. You have to check if reagents in inclusors are ok or over passed, I would change all reagents. I also have noticed some formalin pigment: your formol is not well tamponated 2009/4/3 Randolph-Habecker, Julie jhabe...@fhcrc.org Folks, I need some input on a problem we're seeing with some mouse brain samples. The samples are from new born mice P0 to P14 which have been fixed in 10% NBF (Fisher brand and well within expiration date) for 72 hours. They are then transferred to 70% etoh and processed on an 8 hour process. After the tissue is embedded, we are taking sagital sections, drying them overnight, and then baking overnight at 60C. They are then stained with HE. This has been working great but now all of a sudden we have very light HE staining, nuclear bubbling, and extensive tissue cracking. I also have noticed some formalin pigment. My first thought is that there was difference in formalin or time in formalin but that is not the case. I also wondered if the tissue might be exposed to excessive heat. We checked all of the temperatures in our processor, embedding center, and water baths - all were within tolerance. Any ideas what might cause all three artifacts? Could it be from inadequate paraffin infiltration? Any help would be greatly appreciated! Thanks, Julie Julie Randolph-Habecker, Ph.D. Staff Scientist - Director Experimental Histopathology Shared Resource Fred Hutchinson Cancer Research Center 1100 Fairview Ave, N. DE-360 (Please note new location) Seattle WA 98109-1024 206-667-6119 jhabe...@fhcrc.org ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Ana Resendes DVM, MSc, PhD Veterinary pathologist Centro de Investigação de Recursos Naturais (CIRN) Secção de Anatomia e Taxonomia Zoológicas, Departamento de Biologia, Universidade dos Açores. Rua da Mãe de Deus, 58 - Apartado 1422 P - 9501-801 Ponta Delgada (Açores) Portugal Tel. (+351) 296 650 000 ext. 1109 Fax (+351) 296 650 100 http://www.uac.pt/~pherg/ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Ana Resendes DVM, MSc, PhD Veterinary Pathologist Centro de Investigação de Recursos Naturais (CIRN) Secção de Anatomia e Taxonomia Zoológicas, Departamento de Biologia, Universidade dos Açores. Rua da Mãe de Deus, 58 - Apartado 1422 P - 9501-801 Ponta Delgada (Açores) Portugal Tel. (+351) 296 650 000 ext. 1109 Fax (+351) 296 650 100 http://www.uac.pt/~pherg/ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Problems with mouse brains
2009/4/3 Randolph-Habecker, Julie jhabe...@fhcrc.org Folks, I need some input on a problem we're seeing with some mouse brain samples. The samples are from new born mice P0 to P14 which have been fixed in 10% NBF (Fisher brand and well within expiration date) for 72 hours. They are then transferred to 70% etoh and processed on an 8 hour process. After the tissue is embedded, we are taking sagital sections, drying them overnight, and then baking overnight at 60C. They are then stained with HE. This has been working great but now all of a sudden we have very light HE staining, nuclear bubbling, and extensive tissue cracking. I also have noticed some formalin pigment. My first thought is that there was difference in formalin or time in formalin but that is not the case. I also wondered if the tissue might be exposed to excessive heat. We checked all of the temperatures in our processor, embedding center, and water baths - all were within tolerance. Any ideas what might cause all three artifacts? Could it be from inadequate paraffin infiltration? Any help would be greatly appreciated! Thanks, Julie Julie Randolph-Habecker, Ph.D. Staff Scientist - Director Experimental Histopathology Shared Resource Fred Hutchinson Cancer Research Center 1100 Fairview Ave, N. DE-360 (Please note new location) Seattle WA 98109-1024 206-667-6119 jhabe...@fhcrc.org ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Ana Resendes DVM, MSc, PhD Veterinary Pathologist Centro de Investigação de Recursos Naturais (CIRN) Secção de Anatomia e Taxonomia Zoológicas, Departamento de Biologia, Universidade dos Açores. Rua da Mãe de Deus, 58 - Apartado 1422 P - 9501-801 Ponta Delgada (Açores) Portugal Tel. (+351) 296 650 000 ext. 1109 Fax (+351) 296 650 100 http://www.uac.pt/~pherg/ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Problems with mouse brains
Folks, I need some input on a problem we're seeing with some mouse brain samples. The samples are from new born mice P0 to P14 which have been fixed in 10% NBF (Fisher brand and well within expiration date) for 72 hours. They are then transferred to 70% etoh and processed on an 8 hour process. After the tissue is embedded, we are taking sagital sections, drying them overnight, and then baking overnight at 60C. They are then stained with HE. This has been working great but now all of a sudden we have very light HE staining, nuclear bubbling, and extensive tissue cracking. I also have noticed some formalin pigment. My first thought is that there was difference in formalin or time in formalin but that is not the case. I also wondered if the tissue might be exposed to excessive heat. We checked all of the temperatures in our processor, embedding center, and water baths - all were within tolerance. Any ideas what might cause all three artifacts? Could it be from inadequate paraffin infiltration? Any help would be greatly appreciated! Thanks, Julie Julie Randolph-Habecker, Ph.D. Staff Scientist - Director Experimental Histopathology Shared Resource Fred Hutchinson Cancer Research Center 1100 Fairview Ave, N. DE-360 (Please note new location) Seattle WA 98109-1024 206-667-6119 jhabe...@fhcrc.org ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
