Re: [Histonet] need help staining 120um human whole brain sections!
Good morning Rene, I have worked out special staining IHC protocols to work on celloidin cut sections from 60um to 600um thick. These are free-floating human whole brainstem & whole brain sections. The IHC is amazing using single, double & triple. We save on antibodies, ABC, polymer, TSA using the parafilm method. For example: 1) I use a sheet of parafilm slightly larger than the section to be stained & it’s placed on flat surface (glass slide or dish or whatever). 2) Using a pipette, small drops of working diluted antibody are placed on the parafilm surface to match the size of the tissue section (from 500ul to 1ml). 3) Then the tissue section is free-floated from PBST/2% Triton onto another cut sheet of parafilm, where carefully the section is floating onto the middle surface of the antibody drops by using a brush. As the section lays flat, the drops spread throughout the tissue surface - evenly covering the bottom of the tissue section. 4) This step is repeated for the top surface & the section is gently sealed by placing another parafilm sheet on the top surface of the section to spread the antibody & seal the section for incubation overnight. This method works, although time consuming! The problem is for the washes, quenching & blocking - we need a semi automatic IHC system to take of these steps. To bad there isn’t a robotic arm we could have built & programed to do the above steps. We need an inventor type person to build what I can imagine. best Maria > On Mar 20, 2017, at 6:51 AM, Rene J Buesa wrote: > > You have a special project → special tasks so your approach has to be equally > special. > Large brain sections are usually stained while floating but for IH with > different and successive steps requiring very expensive reagents, floating > sections is not well suited. > You should affix the sections to large slides, and I imagine will not be > brain whole sections but limited to some areas. > In that case for IHC you can stain several sections in a humid chamber, > manually. I do not imagine an automatic system for this task. > It will be a costly and slow process indeed. > René > > > On Sunday, March 19, 2017 8:27 PM, Maria Mejia via Histonet > wrote: > > > > Or lab is currently processing a human whole brain. In about a month or two, > the whole brain, which will be encased > in celloidin & serial sections will be cut at 120um each. Now, we’ve bought > an old Tetrander cast iron microtome. If > you haven’t seen one of these microtomes, I can tell you it’s BIG! > > Now, we’ll have to stain quite a number of these sections for IHC. In fact > too many to handle manually. If possible, need > to find a way to at least stain the majority of these sections in a > semi-automatic system e.g. washes, quenching & blocking. > > Does any one think it’s possible to convert a LABGO processor (made in India > & can hold 2 liter glass beakers) or a > Sakura-Tissue-Tek 4640 (also holds glass beakers - both are old style > circular processors using glass beakers for staining > these sections? Does anyone have an alternative system? I could sure use some > input or ideas anyone welcome > and most appreciated. > > Maria Mejia > Lead Histologist > UCSF > Mission Bay > San Francisco, CA > > > > > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu <mailto:Histonet@lists.utsouthwestern.edu> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> > > ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] need help staining 120um human whole brain sections!
Or lab is currently processing a human whole brain. In about a month or two, the whole brain, which will be encased in celloidin & serial sections will be cut at 120um each. Now, we’ve bought an old Tetrander cast iron microtome. If you haven’t seen one of these microtomes, I can tell you it’s BIG! Now, we’ll have to stain quite a number of these sections for IHC. In fact too many to handle manually. If possible, need to find a way to at least stain the majority of these sections in a semi-automatic system e.g. washes, quenching & blocking. Does any one think it’s possible to convert a LABGO processor (made in India & can hold 2 liter glass beakers) or a Sakura-Tissue-Tek 4640 (also holds glass beakers - both are old style circular processors using glass beakers for staining these sections? Does anyone have an alternative system? I could sure use some input or ideas anyone welcome and most appreciated. Maria Mejia Lead Histologist UCSF Mission Bay San Francisco, CA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] need automation system for fluorescent Multiplex IHC
I forgot to mention in my email below our sections will be paraffin for Multiplex fluorescent. Thank you > On Feb 10, 2017, at 10:43 AM, Maria Mejia wrote: > > Dear All, > > I work in a research lab where the focus is on early stages of Alzheimer’s > Disease. > We are seriously considering using an automated staining system for > fluorescent Multiplex > IHC, for the simultaneous detection of multiple (2-6 or more) proteins of > interest in > FFPE brain sections. > > However, our lab has never used an automated staining system before, so we > lack > the abilities and experience to make the correct choice for our lab’s needs. > > Cost; the price of a USED system, since we are a research lab since a new > system is more > expensive, user friendly & size of system are of course major considerations > as well > as the consumables that go along with the system e.g. reagents, containers, & > anything else > to consider as well. > > Please, I’m reaching out to anyone (system users & vendors) who can help our > lab make a smart choice. > I would be most grateful for any recommendations or suggestions. I very much > look forward to hearing > from you. > > Best > Maria Mejia > Lead Histologist > UCSF > Memory & Aging Center > > ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] need automation system for fluorescent Multiplex IHC
Dear All, I work in a research lab where the focus is on early stages of Alzheimer’s Disease. We are seriously considering using an automated staining system for fluorescent Multiplex IHC, for the simultaneous detection of multiple (2-6 or more) proteins of interest in FFPE brain sections. However, our lab has never used an automated staining system before, so we lack the abilities and experience to make the correct choice for our lab’s needs. Cost; the price of a USED system, since we are a research lab since a new system is more expensive, user friendly & size of system are of course major considerations as well as the consumables that go along with the system e.g. reagents, containers, & anything else to consider as well. Please, I’m reaching out to anyone (system users & vendors) who can help our lab make a smart choice. I would be most grateful for any recommendations or suggestions. I very much look forward to hearing from you. Best Maria Mejia Lead Histologist UCSF Memory & Aging Center ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Unprocessing tissue from paraffin
Hi Millis, Lets see if I’m understanding you correctly. 1) You have unprocessed tissue. What kind of unprocessed tissue & how thick are the free-floating sections? 2) Are the tissue fixed? If so, with what? & For how long? 3) You want to stain the unknown whole mount using various histochemical stains or IHC stains? 4) Can you please explain why after staining you need to reprocess for paraffin? Cause once these mystery sections are processed you'll have to cut them using a rotary microtome. I can maybe help you, but your going to have to provide more detail information. Also provide your processing protocol. Regards Maria Mejia Lead Histologist Memory & Aging Center UCSF > On Sep 21, 2016, at 12:38 PM, Caroline Miller via Histonet > wrote: > > Hi there Histonetters! > > I am currently working through some issues of unprocessing tissue to then > restain 'whole mount' with various stains, and then reprocess back to > paraffin. > > I am running into issues of tissue being brittle and scratchy tissue after > reprocessing and sectioning. > > Can anyone offer any advice on kinder dewaxing procedures and / or > additives to the unprocessing / processing fluids that might help in the > resulting piece of tissue? > > I have currently been putting it through the cleaning cycle on the > processor, so standard xylene then alcohol treatment. > > thanks in advance for your advice! > mills > > > > > -- > Caroline Miller (mills) > Director of Histology > 3Scan.com > 415 2187297 > ___ > 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] slide labeler advice for research lab
Our research lab is in the market for a slide labeler. We are an IHC research lab (NOT clinical) working on both paraffin & thick free-floating human whole brain & brainstem sections. Since the lab has never used a slide labeler before (we’ve been manually making our labels), the labeler should be easy to use, so no one has to spend a lot of time figuring & programming the unit. Since we use paraffin standard glass slides as well as 2 inche x 3 inch glass slides, we need a labeler that we could create formats for different sample sizes and types e.g. ID #, Casp 6 + TH IHC + (name of counterstain used) & date..etc. The unit should also have barcoding capabilities. Any advice or suggestions are appreciated. Vendors welcome also. Maria Mejia Lead Histologist Memory & Aging Center UCSF SF CA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC!
Happy New Year Everyone, I'm the lead histologist working in an IHC based research lab focused on early stages of Alzheimer's disease. We work on paraffin sections processed & cut from 600um celloidin sections. Including a lot of 60um cellodin sections from whole human brainstem. For years, everything has going good regarding counterstaining after single & double IHC staining on 60um free-floating sections. However for the past two months we've struggled to achieve good visible counterstaining on IHC sections to count the stained neurons - to see clearly the nucleus & nucleolus! For a number of years, Gallocyanine was our choice of counterstain after IHC. Now, it's NOT working (neurons not stained visible enough to count). We've also tried cresyl violet counterstain - staining too weak! In both counterstains, we modified the staining protocols quite a number of times to get good visible staining - nothing!!! Strangle because we get lovely counterstained neurons with NO IHC staining on our 60um sections, but as soon as we take the sections through the IHC protocol e.g. antigen retrieval, antibodies & chromogens - counterstain is too weak! Our paraffin IHC sections work & look wonderful! Now, my PI wants to try methyl green counterstain, however I think we'll have the same problem. Here's what I need help with: 1) Can someone please explain the reason or theory behind the failure of counterstain uptake by cells such as human neurons on 60um celloidin sections? 2) Can anyone please offer staining protocols that use alternative dehydration & clearing reagents. I've been using alcohols dehydration (96% & 100%) without success as well as clearing with xylene - which hardens the tissue if left too long in this reagent. I was thinking of perhaps using acetone instead of alcohols & maybe using a methyl salicylate or chloroform. Thoughts anyone? I wish Dr Chris van der Loos was still with us. I'd dearly like to hear from anyone who can help with this issue - Gayle Callis, Terry Johnson, Dr Hohn Kiernan et al. Any assistance anyone can provide will be greatly appreciated! Best Maria Mejia UCSF Memory & Aging Department San Francisco, CA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet