Hello.
I am a beginner researcher experimenting with IHC for the first time.
I would like to know how to make sure it is completely deparaffinized when it is fixed with paraffin and removed with xylene.
Or is there any reason not to check?
I am not a major, I am immature ...
If you know the related contents, I would really appreciate it if you send me an attached file.
There is no way to visualize deparafinization from within a tissue section with naked eye. However, dipping in pure xylene I and xylene II for 3 minutes each ensures visible deparafinization from the surrounding of the tissue sections.
Since you are a beginner, you may want to read this book for a reference:
Bancroft, J.D. and Gamble, M. (2008) Theory and Practice of Histological Techniques. 6th Edition, Churchill Livingstone, Elsevier, China.
Best of luck
The section should be translucend after deparaffination in xylene.
Try to go very quick through xylene into the 100% and 96% ethanol. Then you will see white smears that are due to paraffinresidues.
Going back to xylene will clear the slide and section.
If deparaffination is really insufficient water doesn't rinse smooth on the surface of the section and shows tiny "bubbles".
Usually 2x 3-5 min in xylene is sufficient for 2-4 µm sections. Dipping is of advantage. Xylene may be reused.
If you are uncertain if deparaffination is sufficient, prolong the duration or add another portion of xylene. This doesnt interfere with IHC staining.
Hi,
I agree with Gudrun Lang , you will notice when the section still has paraffin.
You can try with longer xylene baths. We have used up to 10 minutes of xylene (x2) and it worked well.
Good luck.
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