I've been trying to make 5 uM sections of injured mouse spinal cords and stain the tissue with Luxol Fast Blue and H&E. At first I was removing the bones from around the spinal cord, but the tissue just fell apart after I got it on the slide. I think because it had been severely damaged. enter image description here

I thought I might be able to hold the tissue together better if I kept the bone, so I took a spine and decalcified it with 500mM EDTA for 1 week and tried again. But the bone tissue just pulled away from the spinal cord. enter image description here enter image description here

These were some of my better images. I have many others that are even less intact. I see the tissue fall apart on the slide during the drying step, after I put the section on the surface of a water bath and catch it on the slide. I have seen some sections spread all the way across the slide. I suspect it has something to do with water getting trapped under the paraffin section and not drying correctly.

How can I improve the procedure to keep the tissue in 1 piece?

  • $\begingroup$ What is your dehydration protocol? What are you using for a fixative? How experienced are you with histology generally? Brain and spinal cord can be tricky tissues to work with. My initial bet is on the dehydration being incomplete. Also a nice attempt on the decalcification and using the bone as a scaffold - I don't have a good sense on whether this is likely to cause other problems, but it seems most typical for people to dissect the cord out rather than keep it in situ. And as you noticed, it's going to be hard to keep the tissue anchored within the bone after the shrinkage of fixation. $\endgroup$
    – Bryan Krause
    Jul 21 '17 at 15:53
  • $\begingroup$ @BryanKrause Fixation is done by transcardial perfusion of 10 mL PBS followed by 10 mL of 4% PFA. The spines are removed and stored in 4% PFA for 1-3 days, then 500 mM EDTA for 1 week, then back into 4% PFA until I have time for paraffin embedding, usually just overnight. I need to double check the dehydration protocol, it's done by a machine with multiple buckets and I was just told to put the samples in the basket and hit start. I'm pretty sure it's 70%, 90%, then 100% Dryzol followed by paraffin, but not sure about the time for each step. Total time is 24 hours. $\endgroup$
    – user137
    Jul 21 '17 at 17:00
  • $\begingroup$ @BryanKrause I have no experience with histology. My advisor just sort of dropped me into a project a year ago and expected me to learn animal surgery, gait analysis, histology etc with almost no training or supervision. Needless to say, this has been a slow project. $\endgroup$
    – user137
    Jul 21 '17 at 17:02
  • $\begingroup$ That's why I asked about your experience with histology - I think it's fairly typical to get thrown into a project like that, whereas professional histologists have a ~2-year professional degree program. Not to mention you are working with some of the most difficult tissues. I would try to find a histology expert that works in your department in another lab to help you out a bit (I myself am a novice at best). Your PFA is made fresh, right? No methanol? $\endgroup$
    – Bryan Krause
    Jul 21 '17 at 17:10
  • $\begingroup$ I'm not familiar with "dryzol" but if you are dehydrating in ethanol some people use several sequential baths in absolute ethanol at the end (again, this should really be absolute ethanol, 100% ethanol, not "reagent ethanol" or some similar alcohols mix that has methanol or isopropyl in it). $\endgroup$
    – Bryan Krause
    Jul 21 '17 at 17:13

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