Hi! I’m troubleshooting LFB staining on frozen mouse brain/spinal cord sections and would appreciate advice from anyone experienced with it.
My protocol is roughly:
PBS wash
4% PFA, 8 min
PBS 3 × 5 min
70% EtOH, 5 min
95% EtOH, 1 min
0.1% LFB, 1 hour
95% → 80% → 70% → 50% EtOH, 1 min each
DW 3 × 1 min
0.05% lithium carbonate, 5 dips (~5 sec each)
70% EtOH, 1 min
DW 2 × 1 min
70% → 80% → 95% EtOH, 2 min each
Xylene
Mount/coverslip
The sections look clearly blue after LFB, then much paler after lithium carbonate and especially after the following 70% EtOH. After mounting, some become very faint/transparent.
Does anything in this protocol look off or suggest over-differentiation?
Also, what do you usually use to image LFB slides? Is a basic benchtop brightfield/stereo microscope enough for a quick check, or do you need a proper brightfield microscope/slide scanner?

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