Having a hard time getting a decent slide out of rat spinal cord sections ive mounted in paraffin. There's tiny streaks/tears in the section it self but not the surrounding wax. Here's the protocol i've been following:
50% alcohol: 30 mins
70% alcohol: 30 mins
90% alcohol: 30 mins
100% alcohol: 30 mins x 2
xylene: 30 mins x 2
wax overnight at 65*C
Does anyone have any suggestions that will fix the small tears in the sample?
At first sight the overnight infiltration at 65°C seems to be too long for such tiny specimens. I think one hour should be sufficient. Also the 65°C seem to be too high. If your paraffin has a melting point about 58°C lower the temp to 60-62°C.
If the specimens stay still brittle the times in 100% ethanol and xylene can be reduced.
For better sectioning you can try to soak the trimmed blocks before sectioning with water for a few minutes. not too long, because then swelling can occur. A further tipp is to cut with very sharp knifes and breath at the surface just before taking a section.
If you get a sufficient section, it may be "restored" in the warm waterbath, so let it swim for a minute before mounting.
Just a thought about fixation. Be sure, that fixation-time is sufficient.
If you make sure that inadequate fixation is not the problem, I suggest to extend all ethanol dehydration steps to 1 hour and to add isopropanol step (1x30min) after it.
Also, you should use toluene instead of xylene (2x15 min) because it allows much better paraffin incorporation.
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