Everyone wrote:
Hi Ana, The OCT on the coated slides will not disappear like it does on regular glass slides. It appears to polymerize along with the coating, but it doesn't seem to interfere on the tissue section with the staining. According to Gayle Callis' recommendation, it's best to try using the least amount of coating rather than more. More doesn't always mean better adhesion, and as Nancy Thomas reported usually ends up causing more imaging interference and stain uptake making for a messier slide. Teri Johnson Manager, Clinical Trial Testing Genoptix, Inc., a Novartis company BioPharma 1811 Aston Avenue Carlsbad, CA 92008 USA Ana, We once tried 4x coated slides thinking, like you, that it would deliver a better quality section and would stay on the slide better. Our researchers did not like them at all because of the high level of background staining and fluorescence. We only use 1x now. Nancy -----Original Message----- From: Ana Maluenda [mailto: <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> amaluenda at svi.edu.au] Subject: [Histonet] Cryojane tape-transfer and hydration of sections Hello Histonetters! I was wondering if there is anyone out there who is currently using the CryoJane Tape-transfer system for bone sections. I have been trying to get good sections for mice tibia and femur and often find myself with problems. I spent a good time trying to improve the quality of sections (tried different temperatures, thicknesses and speed as well as 1x vs 4x coated slides). The sections improved (although not perfect), but I was planning to move on for a staining trial. Currently, the sections are taken at 5um, in a 4x coated slide, at -26oC. However, now I get myself with a problem when hydrating the sections. At the moment, once the sections are taken, they are kept in -20oC (for short-storage). They are then taken out and left at RT for 30 min before hydration with PBS for another 30 min. I have noticed that it seems as if the OCT around the sections doesn't completely dissolve. I have already tried PBS vs dH2O and played around with times (from 5 min to overnight) with no differences. Has anyone had this happening before? Can it be because of the 4x coat? Is there anything I can do to? And would this be a problem for immunofluorescence? Any advice would be much appreciated! Thanks in advance, Ana Maluenda **************************************************************************** ****************************************************** To Teri and all, Thanks Teri for reiterating my suggestions along with more information. We found the 4X coating to be unacceptable. It is more sticky but the polymer is too thick and will cause more background when one needs to deal with for immunofluorescence work. With murine turbinates, unfixed and calcified, we did use the 1X and even 1/2X but it was necessary to flash the UV three times, waiting 30 sec between flashes so the capacitor could build up charge. It takes patience. We sectioned at 5 µm but the d profile tungsten carbide knife was very sharp so do not cut on an edge you use for trimming into the block. Careful removal of the pink tape is required, inside cryostat chamber, brace the corner of slide, then pull pink tape diagonally across the section from one corner to opposite corner. You have to play with temperatures with everything the same temperature. For turbinates -30C worked well, but tape, slides, rollers, etc. blade and samples were at that temperature, including the UV platform. Sectioning temperatures vary with different laboratories, the sections can be air dried like any other frozen section destined for solvent fixation. I would go ahead and air dry a frozen section and store at -80C, not -20C since the colder temperature is more suitable for retaining antigenicity. We preferred using fresh, unfixed tissues, snap frozen correctly (not in a cryostat!) over NBF or PFA prefixed/cryoprotected snap frozen bone as we found the fresh tissue frozen section stayed transferred to the slide better. Other may have a different experience. You cannot remove the OCT from the polymerized surface and don't need to do that. There are some things your researchers will have to live with. 1) polymer exists even with 1X, but focusing on the plane of the section and what is in the section will work. 2) make sure you work with the brightest fluorophors i.e. Alexa dyes. 3) autofluorescence is caused by many things and if your sections are prefixed with NBF or PFA, then aldehyde induced auto-fluorescence will happen but can be treated. Go on web and get Autofluorescence causes and cures pdf. 4) if you work with Near infra-red fluorophores, there is no auto-fluorescence in the NIR region but the eye can't see it but the photos are spectacular. 5) Totally fill in those weird polymer gaps, be overly generous with antifade mounting media. i.e. Molecular Probes AntiFade Reagent, ready to use. 6)if working with confocal, you can rule out backgrounds/autofluorescence or with spectral imaging. We learned to ignore some of the polymer background and used only the brightest fluorophores. Alexa dyes and Dylights are two or many available now. What you want is a fluorophore signal is brighter than the goo and or autofluorescence caused by aldehyde fixation. This goo background problem is ongoing and has been a common complaint for years but if you need the undecalcified bone section, you learn to live with it. Sadly, this remains unchanged so we live with some trade offs. We have worked with 1/2X coating with success on murine bone but extra UV flashes had to be done. Turbinates are easier than tibia and femur to transfer, but it takes practice and as said before, patience. Normal hydration occurs but OCT is glued to slide along with section, and never dissolves away. If you find Cryojane to be totally unacceptable, check out this website and how they use the Kawamoto film method that results in some spectacular cryosectioning of bone along with beautiful immunofluorescent staining. Go to this site for the whole methodology outlined in how to detail. http://bonebase.org/histomorphometry This group has some excellent publications too. I hope this helps. Gayle M. Callis HTL/HT/MT(ASCP) _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet