Hi everyone! I tried my first H&E stain with one of the kits I ordered [Vector Labs, cat.no H-3502], and I stained all my slides. However, when I imaged the sections, there was no pink stain only blue (perhaps I did not rinse Hematoxylin enough after staining and it is overpowering the other stain). How would I be able to fix this? I've read in other posts that xylene should get the covers to fall off: how long should I leave the slides in xylene for? would the mounting media washout on its own? and should I start staining from the beginning?
The protocol from Vector Labs is:
1. Deparaffinize sections, if required, and hydrate to distilled water.
2. Apply adequate Hematoxylin to completely cover tissue section and incubate for 5 minutes.
3. Rinse slide in 2 changes of distilled water (15 seconds each) to remove excess stain.
4. Apply adequate Bluing Reagent to completely cover tissue section and incubate for 10-15 seconds.
5. Rinse for slide in 2 changes of distilled water (15 seconds each).
6. Dip slide in 100% ethanol (10 seconds) and blot excess off.
7. Apply adequate Eosin Y Solution to completely cover tissue section and incubate for 2-3 minutes.
8. Rinse slide using 100% ethanol (10 seconds).
9. Dehydrate slide in 3 changes of 100% ethanol (1-2 minutes each).
10. Clear and coverslip.
Thanks!!!
Yes, you can remove the coverslips from the slides. Put the slides into Xylene over night or longer. After the coverslips have fallen down use the following protocol:
Xylene -- 2 x 3 min
Isopropanol -- 3 min
Ethanol 100 % -- 2 x 3 min
Ethanol 70 % -- 3 min
destilled water - short rinse
alcoh. Eosin Y solution -- 2-3 min
destilled water -- short rinse
Ethanol 70 % -- 10 dips
2 x Ethanol 100% -- 10 dips each
Isopropanol -- 2 min
Xylene -- 2 x 3 min
mounting in DPX or similar xylene soluble mounting media
If the haematoxylene stain is to strong you can differentiate it with HCl-alcohol
Analternative to the Bluing Reagent is 5 -10 min runnig tap water
Ute Neubacher Hi there, would my sections de-attach from the slides? They were fixed already to the slide before staining.
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