Re: [Histonet] HELP! Need some old fashioned histology advice
I don't know if the slides were subbed or not, but they looked improvised. The point is, at some point the sections are going to have to be mounted for visualisation, right? Someone or something is going to look at the section under magnification? If you mount them on glass *before* staining, the whole problem of batching becomes one of improvising a giant stain rack and giant staining line. Don't forget, you need giant cover slips, giant slide folders, and giant postdocs. I know that lab glassware is custom ordered every day. Sounds expensive and time consuming to set up, but it beats fiddling around with free floating tissue anyday, IMHO. I don't know what the Ab penetration is going to be like on a 100um section, because they weren't using IHC at Genentech, they were doing in vitro DNA hybridization on the slides. I'm assuming the section thickness is necessary because the PI wants to follow axonal pathways and see the patterns of staining? Sounds like a fun project. If you need more help later you can PM me. Sincerely, Jay A. Lundgren, M.S., HTL (ASCP) On Mon, Jan 5, 2015 at 10:38 PM, Mejia, Mary mary.me...@ucsf.edu wrote: Hello Patsy, Thank you very much for responding! Yes of course, I'll be removing the celloidin from each section - we normally do this on smaller human whole brainstem sections using ether/100% EA 1:1 - 3x - 3 minutes each with agitation. The latter type of sections are very easy to work with, however these future big boys I'll be getting is another thing. Our lab is currently alcohol processing a human whole brain after the last 95% EA - it will be embedded in 2% celloidin placed inside a very large desiccator under 20 psi pressure. This part like every step will take some period of time, I need to test several different IHC methods hope one will actually work. If you have any further ideas or thoughts on this subject - shoot me an email. Thank you again for responding. Maria -- *From:* Patsy Ruegg [prueg...@hotmail.com] *Sent:* Sunday, January 04, 2015 7:03 PM *To:* Jay Lundgren; Maria Mejia *Cc:* Histonet@Lists. Edu; Mejia, Mary *Subject:* RE: [Histonet] HELP! Need some old fashioned histology advice I have done something similar to this but I used tissue that was fixed but not processed and embedded, this is called enblock labeling, I infiltrated the fixed tissue with the IHC reagents, in a vial/tube, the blocking reagents, then the antibody, then the detection reagents and DAB, then dehydrated the tissue. I used vials or tubes on a platform shaker and would infiltrate reagents for days, then after it was done I infiltrated and embedded the tissue in glycol methacrylate (GMA) so that I could section it, it actually worked. The tissue was already IHc LABeled so all I did to the 5 micron sections after they were cut was a hematoxylin counterstain, this was mineralized bone so I had to embedd in something hard like GMA to section. Will you remove the Celloidin before trying to do the IHC staining? 100 micron sections might be easy to float/handle using a glass pipette for transferring. Sounds like an interesting project, good luck and feel free to ask for advise and keep us posted on your progress. Cheers, Patsy Patsy Ruegg, HT(ASCP)QIHC Ruegg IHC Consulting 40864 E Arkansas Ave Bennett, CO 80102 H 303-644-4538 C 720-281-5406 prueg...@hotmail.com Date: Sun, 4 Jan 2015 11:58:38 -0600 From: jaylundg...@gmail.com To: mbmph...@gmail.com Subject: Re: [Histonet] HELP! Need some old fashioned histology advice CC: histonet@lists.utsouthwestern.edu; mary.me...@ucsf.edu I can help with the old fashioned advice: - 1 scant teaspoon simple syrup - 2 dashes Angostura Bitters, plus more to taste - 1 half dollar–sized slice orange peel, including pith - 2 ounces good-quality rye or bourbon - 1 maraschino cherry As for the Histology, is there any reason you cannot mount the sections onto glass slides? When I was working at Genentech they were cutting frozen sections through whole rabbits and mounting the sections on (giant) glass slides. I think that rolling the tissue up, inserting it, and then removing it from a glass tube would destroy the tissue. Sincerely, Jay A. Lundgren, M.S., HTL (ASCP) On Sat, Jan 3, 2015 at 8:01 PM, Maria Mejia mbmph...@gmail.com wrote: First, the very best of holidays to everyone. Now for the histology part. Our lab's focus is on the early stages of Alzheimer's Disease in the Brainstem using celloidin processing embedding for IHC staining. This year, our lab will be receiving 6 post-mortem whole human brains (1 every other month). After fixation, processing celloidin embedding, the whole brain will be serially cut at 100um thick. Each brain section will be 5
RE: [Histonet] HELP! Need some old fashioned histology advice
Hello Patsy, Thank you very much for responding! Yes of course, I'll be removing the celloidin from each section - we normally do this on smaller human whole brainstem sections using ether/100% EA 1:1 - 3x - 3 minutes each with agitation. The latter type of sections are very easy to work with, however these future big boys I'll be getting is another thing. Our lab is currently alcohol processing a human whole brain after the last 95% EA - it will be embedded in 2% celloidin placed inside a very large desiccator under 20 psi pressure. This part like every step will take some period of time, I need to test several different IHC methods hope one will actually work. If you have any further ideas or thoughts on this subject - shoot me an email. Thank you again for responding. Maria From: Patsy Ruegg [prueg...@hotmail.com] Sent: Sunday, January 04, 2015 7:03 PM To: Jay Lundgren; Maria Mejia Cc: Histonet@Lists. Edu; Mejia, Mary Subject: RE: [Histonet] HELP! Need some old fashioned histology advice I have done something similar to this but I used tissue that was fixed but not processed and embedded, this is called enblock labeling, I infiltrated the fixed tissue with the IHC reagents, in a vial/tube, the blocking reagents, then the antibody, then the detection reagents and DAB, then dehydrated the tissue. I used vials or tubes on a platform shaker and would infiltrate reagents for days, then after it was done I infiltrated and embedded the tissue in glycol methacrylate (GMA) so that I could section it, it actually worked. The tissue was already IHc LABeled so all I did to the 5 micron sections after they were cut was a hematoxylin counterstain, this was mineralized bone so I had to embedd in something hard like GMA to section. Will you remove the Celloidin before trying to do the IHC staining? 100 micron sections might be easy to float/handle using a glass pipette for transferring. Sounds like an interesting project, good luck and feel free to ask for advise and keep us posted on your progress. Cheers, Patsy Patsy Ruegg, HT(ASCP)QIHC Ruegg IHC Consulting 40864 E Arkansas Ave Bennett, CO 80102 H 303-644-4538 C 720-281-5406 prueg...@hotmail.com Date: Sun, 4 Jan 2015 11:58:38 -0600 From: jaylundg...@gmail.com To: mbmph...@gmail.com Subject: Re: [Histonet] HELP! Need some old fashioned histology advice CC: histonet@lists.utsouthwestern.edu; mary.me...@ucsf.edu I can help with the old fashioned advice: - 1 scant teaspoon simple syrup - 2 dashes Angostura Bitters, plus more to taste - 1 half dollar–sized slice orange peel, including pith - 2 ounces good-quality rye or bourbon - 1 maraschino cherry As for the Histology, is there any reason you cannot mount the sections onto glass slides? When I was working at Genentech they were cutting frozen sections through whole rabbits and mounting the sections on (giant) glass slides. I think that rolling the tissue up, inserting it, and then removing it from a glass tube would destroy the tissue. Sincerely, Jay A. Lundgren, M.S., HTL (ASCP) On Sat, Jan 3, 2015 at 8:01 PM, Maria Mejia mbmph...@gmail.com wrote: First, the very best of holidays to everyone. Now for the histology part. Our lab's focus is on the early stages of Alzheimer's Disease in the Brainstem using celloidin processing embedding for IHC staining. This year, our lab will be receiving 6 post-mortem whole human brains (1 every other month). After fixation, processing celloidin embedding, the whole brain will be serially cut at 100um thick. Each brain section will be 5 inches x 4.5 inches in size. I will given 250 of these whole brain sections to stain for tau IHC...that's 1500 whole brain sections/year!!! 1) Does anyone have experience doing manual IHC staining of large free-floating brain sections? 2) What type of staining tools, dishes or other essential equipment can anyone recommend? 3) What's the most efficient way to stain 250 sections for batch IHC staining - such as transferring batch sections (maybe 5-10) from reagent to reagent? 4) What type of batch apparatus to use? As for the antibody ABC steps, I was thinking of placing each section inside a large glass cigar tube (yep, people use large glass tubes with fitted cap to store cigars), with 5ml of antibody or ABC reagent gently agitate on a shaker/rotator at room temp during the incubation. Does anyone have ideas on this? Please, any ideas, suggestions or recommendation anyone can provide will be most greatly appreciated. Best regards Maria Mejia UCSF Department of Neurology San Francisco, CA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http
Re: [Histonet] HELP! Need some old fashioned histology advice
I can help with the old fashioned advice: - 1 scant teaspoon simple syrup - 2 dashes Angostura Bitters, plus more to taste - 1 half dollar–sized slice orange peel, including pith - 2 ounces good-quality rye or bourbon - 1 maraschino cherry As for the Histology, is there any reason you cannot mount the sections onto glass slides? When I was working at Genentech they were cutting frozen sections through whole rabbits and mounting the sections on (giant) glass slides. I think that rolling the tissue up, inserting it, and then removing it from a glass tube would destroy the tissue. Sincerely, Jay A. Lundgren, M.S., HTL (ASCP) On Sat, Jan 3, 2015 at 8:01 PM, Maria Mejia mbmph...@gmail.com wrote: First, the very best of holidays to everyone. Now for the histology part. Our lab's focus is on the early stages of Alzheimer's Disease in the Brainstem using celloidin processing embedding for IHC staining. This year, our lab will be receiving 6 post-mortem whole human brains (1 every other month). After fixation, processing celloidin embedding, the whole brain will be serially cut at 100um thick. Each brain section will be 5 inches x 4.5 inches in size. I will given 250 of these whole brain sections to stain for tau IHC...that's 1500 whole brain sections/year!!! 1) Does anyone have experience doing manual IHC staining of large free-floating brain sections? 2) What type of staining tools, dishes or other essential equipment can anyone recommend? 3) What's the most efficient way to stain 250 sections for batch IHC staining - such as transferring batch sections (maybe 5-10) from reagent to reagent? 4) What type of batch apparatus to use? As for the antibody ABC steps, I was thinking of placing each section inside a large glass cigar tube (yep, people use large glass tubes with fitted cap to store cigars), with 5ml of antibody or ABC reagent gently agitate on a shaker/rotator at room temp during the incubation. Does anyone have ideas on this? Please, any ideas, suggestions or recommendation anyone can provide will be most greatly appreciated. Best regards Maria Mejia UCSF Department of Neurology San Francisco, CA ___ 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] HELP! Need some old fashioned histology advice
I have done something similar to this but I used tissue that was fixed but not processed and embedded, this is called enblock labeling, I infiltrated the fixed tissue with the IHC reagents, in a vial/tube, the blocking reagents, then the antibody, then the detection reagents and DAB, then dehydrated the tissue. I used vials or tubes on a platform shaker and would infiltrate reagents for days, then after it was done I infiltrated and embedded the tissue in glycol methacrylate (GMA) so that I could section it, it actually worked. The tissue was already IHc LABeled so all I did to the 5 micron sections after they were cut was a hematoxylin counterstain, this was mineralized bone so I had to embedd in something hard like GMA to section. Will you remove the Celloidin before trying to do the IHC staining? 100 micron sections might be easy to float/handle using a glass pipette for transferring. Sounds like an interesting project, good luck and feel free to ask for advise and keep us posted on your progress. Cheers, Patsy Patsy Ruegg, HT(ASCP)QIHC Ruegg IHC Consulting 40864 E Arkansas Ave Bennett, CO 80102 H 303-644-4538 C 720-281-5406 prueg...@hotmail.com Date: Sun, 4 Jan 2015 11:58:38 -0600 From: jaylundg...@gmail.com To: mbmph...@gmail.com Subject: Re: [Histonet] HELP! Need some old fashioned histology advice CC: histonet@lists.utsouthwestern.edu; mary.me...@ucsf.edu I can help with the old fashioned advice: - 1 scant teaspoon simple syrup - 2 dashes Angostura Bitters, plus more to taste - 1 half dollar–sized slice orange peel, including pith - 2 ounces good-quality rye or bourbon - 1 maraschino cherry As for the Histology, is there any reason you cannot mount the sections onto glass slides? When I was working at Genentech they were cutting frozen sections through whole rabbits and mounting the sections on (giant) glass slides. I think that rolling the tissue up, inserting it, and then removing it from a glass tube would destroy the tissue. Sincerely, Jay A. Lundgren, M.S., HTL (ASCP) On Sat, Jan 3, 2015 at 8:01 PM, Maria Mejia mbmph...@gmail.com wrote: First, the very best of holidays to everyone. Now for the histology part. Our lab's focus is on the early stages of Alzheimer's Disease in the Brainstem using celloidin processing embedding for IHC staining. This year, our lab will be receiving 6 post-mortem whole human brains (1 every other month). After fixation, processing celloidin embedding, the whole brain will be serially cut at 100um thick. Each brain section will be 5 inches x 4.5 inches in size. I will given 250 of these whole brain sections to stain for tau IHC...that's 1500 whole brain sections/year!!! 1) Does anyone have experience doing manual IHC staining of large free-floating brain sections? 2) What type of staining tools, dishes or other essential equipment can anyone recommend? 3) What's the most efficient way to stain 250 sections for batch IHC staining - such as transferring batch sections (maybe 5-10) from reagent to reagent? 4) What type of batch apparatus to use? As for the antibody ABC steps, I was thinking of placing each section inside a large glass cigar tube (yep, people use large glass tubes with fitted cap to store cigars), with 5ml of antibody or ABC reagent gently agitate on a shaker/rotator at room temp during the incubation. Does anyone have ideas on this? Please, any ideas, suggestions or recommendation anyone can provide will be most greatly appreciated. Best regards Maria Mejia UCSF Department of Neurology San Francisco, CA ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] HELP! Need some old fashioned histology advice
First, the very best of holidays to everyone. Now for the histology part. Our lab's focus is on the early stages of Alzheimer's Disease in the Brainstem using celloidin processing embedding for IHC staining. This year, our lab will be receiving 6 post-mortem whole human brains (1 every other month). After fixation, processing celloidin embedding, the whole brain will be serially cut at 100um thick. Each brain section will be 5 inches x 4.5 inches in size. I will given 250 of these whole brain sections to stain for tau IHC...that's 1500 whole brain sections/year!!! 1) Does anyone have experience doing manual IHC staining of large free-floating brain sections? 2) What type of staining tools, dishes or other essential equipment can anyone recommend? 3) What's the most efficient way to stain 250 sections for batch IHC staining - such as transferring batch sections (maybe 5-10) from reagent to reagent? 4) What type of batch apparatus to use? As for the antibody ABC steps, I was thinking of placing each section inside a large glass cigar tube (yep, people use large glass tubes with fitted cap to store cigars), with 5ml of antibody or ABC reagent gently agitate on a shaker/rotator at room temp during the incubation. Does anyone have ideas on this? Please, any ideas, suggestions or recommendation anyone can provide will be most greatly appreciated. Best regards Maria Mejia UCSF Department of Neurology San Francisco, CA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
