Hello,
I am staining paraffin sections (10 microns) with Alcian blue and Nuclear Fast
Red as standard protocol as below.
The problem is that I see some sections stained with alicain blue show some
variability (hope i am able to attach image);
alcian blue stain are washed out in some sections even in the same slide. Those
sections show pink color in the cartilage.
Clearing
dehydrate
1% Alcain blue (PH2.5) 30 mins
dip in tap water
running water 2mins
dip deionized water
0.1% Nuclear Fast red 5 mins
dip deionized water 30 sec X 2
95% ethanol 1min
100% ethanol 1 mins X 2
clearing
Is this something to do with filtering alcian blue or nuclear fast red?
I filtered alcian blue using needles to eliminate a big undisolved particle,
but I did not filter the nuclear fast red since it seems all dissolved well.
also I rarely had the problem last time i used the same staining.
I know i need to filter with filter paper but when i tried, even with non-fine
filter paper, it absorbed too much dye and was not successful.
I tried to wash nuclear fast red with running tap water 1 min and dip in
deionized water intead of twice wash of deionized water as described above.
However, variable staining of alcian blue still present.
Does anybody have same problem before?
I appreciate any suggestion to solve this.
Thanks.
Rui
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