[Histonet] Embedding paraffin for brain tissue?
Hi Everyone: In November, there was a discussion concerning different types of embedding paraffin. Do people find that a certain kind of wax is preferable for embedding brain tissue, or doesn't it matter? I also use Surgipath EM-400, but have decided to try another brand. I have been using this paraffin for about 25 years. It used to cut like butter, with beautiful ribbons and no lines. I would put the blocks on ice, wait 2 hours, and then cut 7-8 cases (about 190 slides) per day easily. Now I am struggling to cut 4-5 cases. I am experiencing a lot of lines in my sections and some rolling and tearing of parts of the sections. This is affecting my lab's productivity, the quality of my sections, and is a source of constant frustration. I am cutting with Surgipath high profile Teflon coated blades. When I tried Thermo-Fisher HP-35 Ultra blades, at first they helped, but soon I experienced rolling of the sections. I have tried changing the angle of the blade, to no avail. So, I thought that I would try a change in the embedding paraffin. Thanks for any suggestions that you may have, Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Immunostaining after prolonged formalin immersion?
Hi Everyone: If tissue has been sitting in 10 % neutral buffered formalin for 3-5 years, can excellent immunostaining still be done on it. I am guessing that some sort of antigen retrieval technique can be done on the tissue, but I am not sure, since we don't do immunostaining ourselves. Thanks, Tim Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Embedding problem
Hi Everyone: When I paraffin embed brain tissue, I gentle push down on all regions (especially the edges) of the specimen(s). It seems that all of the areas of the specimen (or each of several specimens) lie flat on the bottom of the mold. This should ensure that I can quickly obtain a full face when trimming, with a minimum loss of tissue. However, I have found that when I remove the solidified blocks from the mold, parts of the specimen, especially the cortical edges of the tissue, have lifted up from the bottom of the mold. I either have to re-embed the tissue, or trim through more tissue than I would like to obtain a full face for sectioning. Has anyone had this problem? How do people avoid it? Thank you, Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Embedding problems-part 2
Hi again Everyone: Thank you for the suggestions. I use the flat end of an old microtome orientation screw as a tamper. I hold down the various parts of the specimen for around 5 seconds each, but I cannot hold down all areas of the specimen at once, due to the small size of the orientation screw. I have found that since brain tissue is so heterogeneous, certain parts are more suseptible to lifting than others, and I hold those down longer. After I embed the specimen, I put it on the freezing plate, embed a couple of more blocks, then top off the first block with a bit more paraffin, and so forth. Possibly the heat of the extra bit of wax is causing the lifting? Thanks again Tim Wheelock ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Processor dehydration cycles
Hi Tom: I deal exclusively with post-mortum brain tissue, so my situation may not apply to you. I do not use formalin on my processor, since the half brain used for brain-cutting has already been thoroughly fixed. So, I have the luxury of using 30%, 50%, 80%, 95%, then three 100% Isopropanols. They used this protocol when I was at a different neuropathology laboratory. I believe the rational for this was that starting with a lower concentration of alcohol, and then more gradually increasing the concentrations, would reduce the concentration gradient between the cells and the solution, and so avoid strong currents from harming the cells. Also, brain tissue may be more delicate that say prostate, skin or uterus. By the way, I actually do not know whether this rational is correct or not. However, in general, if you can start at 70%, it can't do any harm. Tim Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA (617) 855-359 Tom McNemar wrote: Hello all, I was wondering what most people use as the first reagent after the formalins on their tissue processor? We have always used a sequence of 70%, 80%, 95%, and 100% but is anyone using 80% or even 95% to start their dehydration? Thanks in advance. Tom McNemar, HT(ASCP) Histology Co-ordinator Licking Memorial Health Systems (740) 348-4163 (740) 348-4166 tmcne...@lmhealth.orgmailto:tmcne...@lmhealth.org www.LMHealth.orgfile:///C:\Documents%20and%20Settings\TMCNEMAR\Application%20Data\Microsoft\Signatures\www.LMHealth.org ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Question about sectioning brain tissue at 10-15 microns
Hi Christina: I use Paraplast to infiltrate the formalin fixed brain tissue, but I use Surgipath Embedding Medium from Leica (Catalogue number 3801320) to embed the tissue. (Surgipath does have about the same melting point as the Paraplast) Even the 10 micron sections (for the Congo red stain) tend to curl, but I just guide them off of the disposable blade with a brush or a pick. Hope this helps, Tim Tim Wheelock Harvard Brain Bank McLean Hospital Belmont MA 617-855-3592 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] LFB Lithium Carbonate Differentiator alternative
Hi Cheryl: Yes there is: 1% Hydroquinone 5% Sodium Sulfite in Distilled water This differentiator, which I use in my lab, acts very quickly. For a 5 micron paraffin section, I dip it only twice, quickly, into the differentiator, and then immediately, into 4 changes of tap water, then a staining dish with tap water. Hope this helps, Tim Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA 617-855-3592 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Laboratory SOP
Hi Everyone: I am putting together an SOP for my neuropathology lab. Does anyone know of a template, example, or guide that I could refer to? Thanks, Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Histotechnician Salary
Hi Everybody: I manage a neuropathology laboratory at a brain tissue bank. We are going to be requesting funds from the NIH for a full-time histotechnician to work with me. We would like someone who is HT or HTL certified, has at least 5 years of experience, preferably in neuropathology, and can work independently. However, I have no idea what the salary would be for this kind of position. Could people let me know how much such a position-or a comparable one in a surgical histology lab-pays? Thank you, Tim Wheelock Assistant Director, Neuropathology Laboratory Harvard Brain Tissue Resource Center McLean Hospital Belmont, MA. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Histotechnician Salary 2
Hi again all: I should have mentioned that the position calls for doing classical neuropathology stains, but not IHC. We have our immunostains done by another lab. Thanks, Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] VIP6 Questions
Good morning Everyone: For those of you who have a Sakura VIP6, do you actually use the bulk reservoirs to let the machine automatically rotate the absolute alcohols and xylene? If not, why not? Also do you let the machine automatically rotate the paraffin reservoirs, or do you do this manually, and if so, why? Also, do you find the touch screen graphics easy to use? Have any problems developed with the display itself? Thanks, Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Follow up question for embedding centers
Hi Everyone: I was wondering if anyone has had experience using two other embedding centers that I have received information on? One is the Medite VALIDA. The second is the HistoPro 150. Also, is a stainless steel cold plate and/or work surface susceptible to rust down the road or not? Thanks, Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] TISSUE PROCESSOR FOLLOW-UP QUESTION
Hi All: First, thank you for all your feedback on the processors. I site-visited a VIP6 at a local hospital From my understanding the VIP6 can rotate absolute alcohol and xylene (using the bulk reservoirs), as well as the paraffin stations, but it cannot rotate other concentrations of alcohol based on hydrometer readings. Am I correct in this appraisal? Tim Wheelock Neuropathology Laboratory Harvard Brain Bank McLean Hospital Belmont, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Tissue Processors
Hi Everyone: I am currently evaluating three tissue processors. They are the Sakura VIP6, the Leica ASP 6025, and the Thermo-Fisher Excelsior ES. I was wondering if people could give me their critical opinions and preferences on these three machines. In addition to reliability and ease of use, I am interested in people's experience with tech support, software, or any other factor-positive or negative-that prompted your decision. I currently have a 14 year old Shandon Hypercenter XP. Thank you, Tim Wheelock Neuropathology Laboratory Harvard Brain Bank McLean Hospital Belmont, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Gloves
Hi Pam: I use nitrile gloves that we get from our Central Supply Room when handling xylene and cover-slipping by hand. They are Kimberly-Clark Sterling Nitrile Powder-Free Exam Gloves. I use just a single glove rather than double-gloving, and that seems to do the trick. The glove usually does not break. However, now that you mention it, I wonder if a single layer really totally prevents penetration of xylene or not. Tim Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA 02478 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Bielschowsky questions, Luxol Fast Blue answers, and Protocol Drift.
Hi Everyone: I had a few questions regarding Bielschowsky silver stains. (1) What adhesive (if any) or type of slide do you use for the stain? (2) How do you clean the glassware? (3) When diluting the 40% formaldehyde when making up the developer, do you consider the 40% formaldehyde as 100% and then dilute it down by using 1 part formaldehyde and 9 parts distilled water? Or do you assume the 40% formaldehyde is 40% and then dilute it down using 1 part formaldehyde and 3 parts distilled water? (My protocol may have inadvertently changed from the first method to the second; I am not sure.) By the way, I want to thank everyone for helping me solve the problem of my Luxol Fast Blue staining the myelin too lightly. I discovered that somehow, I had started adding twice the amount of acetic acid to the Luxol staining solution as I should of. (This protocol drift, where a mistake can actually find its way into a written protocol, can be a real problem in a lab, especially when working for years by oneself, as I have) . But I also found that even reducing the acetic acid, while helping a lot, did not completely fix the problem. I needed to switch from staining the tissue for 2 hours at 60C to a full over-night (why I never needed to switch times before is a mystery). That did the trick beautifully. The myelin is staining perfectly again. Thanks again, Tim Wheelock Harvard Brain Bank Belmont, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Hydrometer questions
Hi everyone: I wanted to thank everyone who gave me advice regarding my Luxol Fast Blue problem. First, I am going to compare using a very old LFB bottle (from the 1950s) and a new bottle. Then I will stain some test slides for 2 hours and 24 hours at 60C. (I am having my oven checked this morning by facilities). To find out how much water is really in my 95% ethanol, I am assuming that I need a hydrometer. (I will also be using it to measure the isopropanol concentrations on my tissue processor). Is there a type of hydrometer that labs use for these purposes? Where do you buy yours from? Can the alcohol/water concentrations be read right off of the hydrometer, like temperature is from a thermometer, or do you need to use an equation. I have never used a hydrometer before. Thank you, Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA 617-855-3592 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Pens for writing cassettes
Hi Everyone: I want to thank everyone who responded to my inquiry regarding marking pens a while ago. I had a problem with the Securline Marker2/Superfrost pens fading from my tissue cassettes. All three of the pens that I tried: StatLab, Leica, and the Histotec brands worked really well. There was no fading on the tissue cassettes at all. The writing was as clear and crisp as when they were first written. I also can still use the Securline Marker 2 pens for writing out my slides. Thanks again, Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Luxol Fast Blue problem
Hi again all: Lately, I have had problems with my Luxol Fast Blue myelin stain not being nearly as dark as it should. I am having my oven checked out because I have had problems with it holding a high enough temperature (at least 60C). This seems to one factor in getting the stain to take. But I suspect there may be other factors involved. Has anyone had this sort of problem before? What did you find was the cause and how did you fix the problem? Thanks for any advice you can provide. Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Thank you
Hi Everyone: I want to thank everyone who gave me advice concerning my cassette labeling problem. I am trying out three different types of markers pens: StatLab, Leica, and NewComer Supply Histo-tec brands. We will also make sure to let the ink dry before putting the cassettes into formalin. If we still have a problem, we will experiment with a different cassette. Thanks again. Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA 617-855-3592 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Thank you
Hi All: I want to thank everyone for the advice on xylene-resistant gloves. It was really helpful. Tim Wheelock Tim Wheelock Assistant Director Harvard Brain Tissue Resource Center Instructor In Neuroanatomy 203 Mailman Research Center McLean Hospital Belmont MA 02478 Phone: 617-855-3592 Fax: 617-855-3199 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Gloves for coverslipping?
Hi Everybody: Does anyone know of a xylene-resistant glove that can be used for coverslipping, and that allows the dexterity necessary to coverslip? Thanks, Tim Wheelock Harvard Brain Bank McLean Hospital Belmont, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Shandon Embedding Center Advice--Thank you
Hi everyone: I want to thank all the laboratories, service companies, equipment manufactures, and company representatives who responded to my questions concerning my Shandon Embedding Center. As it turned out, I had the thermal fuse replaced and now my 23 year old machine is up and running again. However, as a result of this experience, I now have some back-up options for buying a new or refurbished machine or renting/leasing one in the future. I will try to put in for a new one next year for our next grant-cycle. Thanks again, Tim Tim Wheelock Assistant Director, Neuropathology Instructor In Neuroanatomy Harvard Brain Tissue Resource Center 203 Mailman Research Center McLean Hospital Belmont MA 02478 Phone: 617-855-3592 Fax: 617-855-3199 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Embedding Center Questions
Hi All: My 23 year old Shandon Embedding Center may be ready to give up the ghost. It has had two repairs of the coolant system for the freezing plate over the last year. Now a thermo-resistor has burnt out, so that the wax reservoir with no long melt the wax. Does anyone know of a company or companies that lease/rent histology equipment? Are there companies who sell used equipment? Also, what do people recommend as far as a new embedding center, in case we can afford it? I am in the Boston area. In the meantime, maybe I can jury-rig something, since it is only the wax reservoir that is down. The cassette holding tank, hot plate, and freezing plate are OK for the moment. Maybe I can melt a few jars of wax, and use a transfer pipet to fill the molds? Any ideas? Thanks, Tim Tim Wheelock Assistant Director, Neuropathology Instructor In Neuroanatomy Harvard Brain Tissue Resource Center 203 Mailman Research Center McLean Hospital Belmont MA 02478 Phone: 617-855-3592 Fax: 617-855-3199 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] 100 micron paraffin sections?
Hi Everyone: A researcher in our building has done a Golgi-Kopse silver stain on some brain tissue. He says that the tissue has come out brittle with this particular Golgi method. His pieces are 3 mm thick and will easily fit inside of a processing cassette. He would like to embed the tissue in paraffin and cut 100 micron sections. Does anyone have a protocol for processing this kind of tissue into paraffin and then sectioning and mounting such thick 100 micron sections? Thanks, Tim Tim Wheelock Assistant Director, Neuropathology Instructor In Neuroanatomy Harvard Brain Tissue Resource Center 203 Mailman Research Center McLean Hospital Belmont MA 02478 Phone: 617-855-3592 Fax: 617-855-3199 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Re: Brain frozen sections
Hi: There is one thing you might try to make frozen brain sections more than acceptable. You can freeze the tissue in liquid nitrogen vapor (LNV) (not directly into liquid nitrogen), let the tissue equilibrate to cryostat temperature, and then cut the sections. The histology is almost as good as a paraffin section. This is assuming that the technique fits the time frame within which you must arrive at a diagnosis. Also we ourselves do not use frozen sections to screen brains diagnostically. We use paraffin sections from the formalin fixed half of the brain. We send the LNV frozen blocks to investigators who cut sections from the frozen blocks for their research. If you would like the details, you can contact one of the following individuals who do this routinely at our brain bank, to see if this protocol is appropriate for your needs. George Tejada 617-855-2646 gtej...@mclean.org Louis Fernandes 617-855-2636 lfernan...@mclean.harvard.edu Good luck. I hope this information helps. Tim Wheelock Assistant Director, Neuropathology Instructor In Neuroanatomy Harvard Brain Tissue Resource Center 203 Mailman Research Center McLean Hospital Belmont MA 02478 Phone: 617-855-3592 Fax: 617-855-3199 Robert Richmond wrote: You guys tell your pathologists to listen to Dr. Hessler - he taught me how to do these preparations, and the basics of interpreting them, when I did a locum tenens for him at the Medical College of Georgia (in Augusta) 6 years ago. I did a lot of them when I got a full time job at a place that did a lot of neurosurgical pathology, a year later. Bob Richmond Samurai Pathologist Knoxville TN * There is nothing you can do to make brain frozen sections acceptable. Your Pathologists need to learn how to read smears, or just accept being wrong 50% of the time. An educated guess based on the imaging is more accurate than frozen sections on intra-axial primary brain tumors. Richard B Hessler, MD Chief of Pathology Erlanger Medical Center Chattanooga, TN ___ 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] Plastic storage containers for brain tissue?
Hi Everyone: We are looking for rectangular plastic containers to store our brain tissue in. We use two sizes, a 60 oz container (for fixed half brains or their slices) and a 128 oz container (for whole fixed brains or their slices). Recently we have been unable to locate a vendor that makes these sizes anymore. Does anyone know of a vendor that still makes these containers? Thanks, Tim Wheelock Harvard Brain Tissue Resource Center 617-855-3592 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
