You wrote:
We have suddenly started losing tissue sections from our Superfrost Plus Slides. Our students have been cutting fixed, frozen, cryosections (20um) and thaw-mounting these onto Superfrost Plus slides. We have suddenly started losing lots of sections from these slides (again!!). The tissue is small - ie cross sections of frog aorta and longitudinal sections of nerves, so any lose of adhesion results in total loss of the tissue. The odd thing is that she is not losing every section on every slide, but half to 3/4 of the sections are falling off within the first rinse. The sections are from the same tissue block on the same slide while some falls and others don't. We are at a loss as to what we can do to rescue these sections. Does anyone know if there is any way to coat the slides in some solution with the tissue on them to help improve the adhesion without losing the ability to do immunofluorescence? Any further advice on cutting / drying protocols are welcomed. This keeps happening and the inconsistency of it has us so frustrated with this that we are thinking of going back to subbing our own slides. Suggestions: 1. It could be a bad lot of slides, however, fixed frozen sections are notorious for falling off slides, even Plus charge. You did not say what fixative you used either? 2. I did not see where you cryoprotect your fixed tissue prior to snap freezing, and this may help, not only to reduce large ice water crystal damage, but also makes sectioning less of a crunchy affair. 3. Dry sections after sectioning in front of a fan at RT, and dry longer. Do NOT store just cut frozens in the cryostat (you didn't say how you handled the section immediately after picking up on the Plus Charge slides. Begin drying at RT immediately after sectioning. 4. Do not touch the surface of slides with fingers, sometime oils from skin transfer to slide surface. 5. It is a waste to money to coat expensive Plus Charge slides and coatings ruin or negate the Plus charge. If you carefully read the slide box insert, you will see this. Do not coat Plus charge slides with anything. If you want to coat slides with a gelatin (protein) subbing solution or Elmers glue (derived from milk products), use regular, clean microscope slides,, coat, air dry and store slides. . 6. Not knowing what or how you rinse, if the rinse is harsh, too fast, it will wash fixed sections off a slide. 7. 20 um is thick, although people do have success with careful and longer drying, depending on the staining method you want to perform. How you thaw this thick section onto a slide may be important. A young man taught us a clever way to do that by mounting section on a cold slide (cryostat chamber temperature, then thawing it an angle for a more gradual melting to surface. (don't put your finger under slide below section so it melts all at once onto the surface). He started at one edge of the section so it thawed from one side to another. He had FLAT sections and no bubbles under a thick fixed section. If your get any unevenness, the section may come up. Picking up a thick section from the blade holder plate may be part of the problem so try his method just for fun and possible success. Dry section well! 8. Fixing the tissue before doing frozen sections compromises the proteins, and once cross linked, tend to not like to stay on Plus charge slides, a common complaint seen on Histonet. Hence, subbing your own slides might be the answer if you had success before. 9. Make sure your blade is sharp so you obtain the flattest section possible. When sectioning, and you did not elaborate - mount first section on slide, lay slide outside cryostat, then pick up the next section next to the first section - we generally put first section closest to label, when picking up, and work down to bottom (away from label) for the rest of the sections if you are performing the mounting from the blade holder plate. Good luck, and be sure to check Histonet Archives for more answers. Gayle M. Callis HTL/HT/MT(ASCP) Bozeman MT _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet