[Histonet] pancreas pH or proteases inhibit LacZ enzyme activity?
Hello, Histonet experts, I have a puzzling bit of data that I am trying to resolve. Our group and another group have performed LacZ stain on mouse pancreas to identify cells expressing a LacZ reporter (the other group has published this expression pattern). I am having trouble with the primary antibody for the protein that LacZ is supposed to report for, so as a backup I tried an antibody for LacZ, which has worked very well for us in other tissues in the past, and has always matched the pattern we see with LacZ stains. With LacZ antibody, I get very robust stain in the islets, (as well as light stain in the surrounding tissue consistent with the LacZ stain pattern) and I see a qualitative dose response to diabetes induction, which makes me think the antibody stain is real. However, we and another group have not seen any LacZ activity in the islets, only in the surrounding tissue. Could the LacZ enzymatic activity be disrupted ex vivo by the pH of the islets themselves, or protease activity in the islets, so we can pick it up by immunostain but not LacZ enzymatic stain? Any other expert opinions as to what's going on? The insulin and glucagon antibody stains are working like champs, beautiful specific signal with very low background. I have been doing LacZ stains on various parts and pieces for the better part of 6 years, this is an unusual case where the confirmation controls didn't match the LacZ stain. And to clarify, I am doing the LacZ (and other) immunostains on pancreas sections that were NOT stained enzymatically for LacZ activity, but are the same genotype. I use Rene's recommended IM embedding protocol for paraffin. On the LacZ side, I stain whole-mount tissue prior to embedding and sectioning, with attention to fixation times and pH of paramount importance. Thanks in advance for your help! Sincerely, Nicole Collette, Ph.D. Lawrence Livermore National Lab collet...@llnl.govmailto:collet...@llnl.gov ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] immunofluorescence mounting medium?
Hello, Esteemed Histonetters, I am trying to get nice publication-quality images of my immunofluorescent tissue sections. I am currently using Prolong Gold, and after I let the stuff cure for several days, my 100X oil-immersion images are still smeary, even after taking into account the unevenness of the tissue section. I don't seem to have this problem with colorimetric stains (histological stains, LacZ, etc.), so I am thinking the mounting medium is part of the problem? It seems to me that it never fully cures at the middle of the coverslip… Does anyone have a recommendation for a mounting medium that works better for this purpose? Any advice is appreciated. Thanks in advance, and Happy Monday! Sincerely, Nicole Collette Lawrence Livermore National Laboratory collet...@llnl.gov ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RNAlater regret
Hello, Esteemed Histonetters! I have a collaborator who asked me to drop his (mouse) bones into RNAlater for RNA isolation, later. These animals were double labeled for histomorphometry as well. He now wants to know if he can back them out of RNAlater and use them for any histology-related protocols? (dynamic histomorphometry, histology stains, immunostains)? Perhaps the best course of action is to back them into fixative and proceed with fingers crossed? If anyone has a better idea or already knows it won't work, it would be much appreciated. Thanks, and Happy Wednesday, Sincerely, Nicole Collette LLNL ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Frost papers on bone labeling
Hello, Esteemed Histonetters, I am wondering if anyone happens to have a 21st century version of a 20th century paper? In Search Of PDF files for any /all of the following: Frost HM. Presence of microscopic cracks in vivo in bone. H. Ford Hosp. Med. Bull. 1960;8:25–35. Frost HM, Villanueva AR, Roth H, Stanisavljevic S. Tetracycline bone labeling. J. New Drugs. 1961;1:206–216. Frost HM. Measurement of human bone formation by means of tetracycline labeling. Can. J. Biochem. Physiol. 1963a;4:31–42. Frost HM. Relation between bone-tissue and cell population dynamics, histology and tetracycline labelling. Clin. Orthop. 1966b;49:65–75. Frost HM. Tetracycline-based analysis of bone remodelling. Calcif. Tissue Res. 1969;3:211–237. My institution a few accessibility issues (umm, like no physical library), and some/all of these are not available online as electronic copies (since they are oldies but goodies). I was hoping to get them sooner than by snail mail through interlibrary loan, and thought it couldn’t hurt to ask. Thanks in advance for your help! Sincerely, Nicole Collette ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Manual embedding
Hi, All, I just saw this question and the responses, thought I would add my own solution to the mix. I do manual embedding, I use a hyb oven for my infiltrating/embedding station (like for hybridizing Southern or Northern blots- who does that anymore?). I have taken out the rotating wheel for spinning bottles, and line the tray at the bottom with foil. Temp control works very well. I have several Wheaton staining boxes (the kind that come with glass inserts for staining 20 slides) that I use for my wax changes for infiltrating, and a couple of metal beakers I use for pouring into molds. I have a little real estate left inside the oven (it's probably around 18x 18 square area) to heat my molds for pouring, and I have a metal heat block in there that you would use for Eppendorf tubes as my forceps warmer. It works pretty well, but does take a long time to heat up those boxes of wax, so I need to plan ahead by about 3 hours. When I pour the molds, I have the oven in front of a drawer under the bench, I line an extra cabinet shelf with foil and lay it over the open drawer to give me some bench space, pour my molds inside the oven and transfer it to the foil-lined shelf to cool so I can move it without the specimen shifting. Then I transfer to a photo tray and cool in the fridge for a couple of hours before releasing the molds. With the door of the hyb oven open I have to embed in shifts and let the molds heat up again, but it works OK. At least it's all contained in one unit. It's hard to do histology in a molecular lab ;) Hope this helps to give a do-it-your-selfer some ideas. Sincerely, Nicole Collette LLNL On 8/2/11 7:53 PM, Scott Parker spar...@vt.edu wrote: Dear Histonetters: I am interested in acquiring a pitcher and heating jacket for melting and pouring paraffin during manual embedding. My work is relatively low volume and in a university research lab setting so I am trying to avoid purchasing an expensive embedding station. Can anyone recommend an honest supplier of used histology equipment that might be able to provide me with this item? Thank you for your expertise! Scott L. Parker ___ 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 processing for undecalcified mouse bones
Hello, All, I am looking into starting a plastic embedding/sectioning endeavor, and am trying to figure out my startup reagents and protocols. I am seeing a lot of protocols for the Technovit system, which uses a slide press to adhere the sections to the slides, and would prefer to avoid expensive startup equipment if possible. I found another protocol that uses silanized slides, water and heat to adhere the sections. Is this comparable to results seen with a slide press, or is there some obvious down-side? I have been mainly looking into MMA/PMMA protocols for my bone, and ideally would like to be able to use the sections for antibody stains in addition to histology stains and mineral assessment- although I will work with the limitations of the medium, I know I can’t always ask for the moon. I will be using adult mouse bones, primarily from appendicular skeleton (long bones). I am trying to start with a relatively do-it-yourself, low throughput option that minimizes startup cost for a system that I may only use short-term. Up until now I have been using either paraffin embedding (decalcified samples), or frozen Cryojane sections (unfixed, undecalcified), but there is potential for plastic to be the best option in some instances. I think I have the sectioning capabilities covered, but would be appreciative of embedding and sectioning protocols (and sage advice from the wise, experienced bone cutters out there, if I’m totally headed in the wrong direction!). A catalog# recommendation for molds/chucks/cassettes (to fit or otherwise adapt to a Leica RM2255 microtome) would be fantastic. Thanks in advance for your help and support. Sincerely, Nicole Collette Lawrence Livermore National Lab collet...@llnl.gov ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Question from one of our researchers
Hi, Teri, How about this? It also gives a brief blurb in the description about commonly used alternatives, if this isn't your thing. I've never tried it, so I can't vouch for it myself, but Invitrogen does make some pretty darn good stuff... http://probes.invitrogen.com/media/pis/mp10045.pdf Sincerely, Nicole Collette Lawrence Livermore National Lab On 12/1/10 9:19 AM, Johnson, Teri t...@stowers.org wrote: Can someone give me ideas to pass along to one of our researchers? Of course adhesion molecule antibodies are the first thought, but not for heterogeneous cell populations. So I was wondering maybe an antibody cocktail? Are there any lectins that might show this? Thanks! I am wondering what kind of cell membrane marker is recommended to stain tissue section for imaging (going to process the image with 3D Imaris software). Cells are mouse tissue section in paraffin and very heterogeneous. To see cell-cell boundary clearly. ___ 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] Blade sharpening service
Hello, esteemed colleagues, I am in need of a service to have my 16cm tungsten carbide Profile D (a.k.a. Sweeney Todd) blades resharpened. Can anyone recommend a service that is relatively accessible to the West coast? I looked in the archives and couldn't find any recent posts, and my Googling has been unproductive. I'd rather not send them back to Leica in Germany to have them sharpened if I don't have to. Thanks for all your help! Sincerely, Nicole Collette UC Berkeley/Lawrence Livermore National Lab ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] strontium vs. calcium in bone
Hi, Everyone, Happy Friday! I was wondering if anyone knows of a histological method to distinguish strontium in bone vs. calcium? My understanding is that mineral stains (von Kossa or Alizarin Red S) do not distinguish, but was wondering if anyone has a procedure I may not have done or seen? I have ways to get at the answer, but for completeness sake, I thought I should ask the experts. Thanks for your help! Sincerely, Nicole Collette LLNL/ UC Berkeley ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Coverslipping Problem - Please Help!
Hi, Brian, It is likely to be a pH issue. If you use BM Purple as your detection chromogen (which is what we use in our lab for whole mounts on X. laevis and X.tropcialis, and mouse embryos for WMISH), then you must keep the embryos at acid pH from BM Purple step onwards, or you will re-activate the BM Purple (hint: Alkaline phosphatase substrate). Light from the microscope compounds your problem. You are likely cryoprotecting and/or coverslipping with neutral or slightly alkaline solution, if you use something acidic, you should be fine. You can adjust the pH of your sucrose pretty readily, so that's not too hard. If you are only coverslipping temporarily for photos (not for archiving), you can simply use a glycerol/PBS pH 5.5 mixture for photos, then discard your slides. Coverslipping for archiving might be a question better addressed my someone else on histonet who is more knowledgeable about mounting media. Good luck! Sincerely, Nicole Collette LLNL/UC Berkeley -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Brian Rabe Sent: Thursday, July 29, 2010 3:14 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Coverslipping Problem - Please Help! Dear Histonet members, I have been cryosectioning Xenopus laevis embryos this summer that have undergone a whole mount chromogenic in situ. The embryos are then fixed overnight in a formaldehyde solution (1X MEMFA) at 4 C, then stored in 1 x PBS until they are cryoprotected in a sucrose solution at least overnight at 4 C. Then they are put in TBS tissue freezing medium for 2-3 hours at room temperature before being frozen and sectioned. The sections come out looking very nice with good morphology, but when they are coverslipped (2 1 minute washes in 1 x PBS to dissolve the tissue freezing medium) with VectaMount AQ aqueous mounting medium, the sections turn very dark under the microscope, especially the gut region. They are so dark that it becomes difficult to see the signal from the in situ. Any help or advice would be GREATLY appreciated! Thank you for your time, Brian Rabe The College of William and Mary ___ 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] Water collecting at bottom of sections
I use FisherBrand SuperFrost Plus charged slides, the only time I have this problem is when my waterbath is too hot, or if for some reason I use warmed slides to retrieve my sections from the bath (like if you lay the unused slides on the edge of the waterbath, or if you use the warm droplet method to spread your sections)? I haven't had much experience with a lot of different slide types/brands though, but it might be an easy fix to the problem you hadn't thought about, as it is more or less unrelated to the brand of slide, although some brands may be more sensitive to this issue... Sincerely, Nicole Collette Lawrence Livermore National Lab/ UC Berkeley -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of nap...@mail.siscom.net Sent: Friday, July 23, 2010 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Water collecting at bottom of sections Hello all, From time to time and depending on what brand of adhesive (or charged) slides I am using, I seem to get a bag of water that drains to the bottom of my sections but doesn't drain out. I have been working in microtomy a long time and have had to deal with this contingency time and time again, but never really have gotten to the bottom of the problem. I spoke with a premium manufacturer of such slides and they seemed to indicate that it is a problem with the coating, but couldn't tell me for sure. All I know is that certain brands do this more than others. If you know what I mean, you know it is a problem. My bath is pure distilled H2O with no gelatin or Sta-on added. It is if the adhesive properties are SO good that they will not release the water when vertically drained and have to be shaken off or cut with a razor blade at bottom to release the water. Anyway, if anyone has an insight or two on this, I would be interested. It seem sthe most challenging issues are ones that seem related to some of the most simple tasks that one has performed for many years!! Manufacturers understand what I mean, but cannot pinpoint the problem for me via phone or e-mail. Anyone see this and have a chemical/mechanical solution they have developed over the years? Thanks! ___ 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] RE: X-gal staining troubleshooting
Hi, Teri, I have seen it in some of my stains, but they were always in embryos of mice from a particular strain (that we did not generate and have incomplete construct information for). I always attributed it to a different LacZ allele, as I have stained litters of a different strain side-by-side with the same reagents on the same day and got the expected blue stain, but it may be something else... If you find out, I'd like to know, too. Sincerely, Nicole Collette LLNL/ UC Berkeley -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Johnson, Teri Sent: Tuesday, June 22, 2010 1:03 PM To: 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] X-gal staining troubleshooting Hi all, I have a question about whole mount x-gal staining. Do any of you have experience with your samples turning brown after incubation with the x-gal? If yes, do you know what causes this and how to keep it from happening? Thanks in advance, Teri Johnson, HT(ASCP)QIHC Managing Director, Histology Facility Stowers Institute for Medical Research Kansas City, MO ___ 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
