Our team is trying to determine the possibility of executing IHC on a slide previously used for histology or vice versa. Thank you :)
This is possible. It is important to mount the sections on adhesive slides. Do a conventional HE-stain. Afterwards you can loose the coverslip again by immerse the slide in xylen till the coverslip can easily be lifted. Go back through xylen, ethanols till deionisized water. Don't let the section dry out. Then start with antigen-retrieval and so on for the IHC (e.g. DAB and nuclear staining with hematoxylin).
The eosin will be solved by antigen-retrieval. Remaining hematoxylin does not interfere with IHC.
This is possible. It is important to mount the sections on adhesive slides. Do a conventional HE-stain. Afterwards you can loose the coverslip again by immerse the slide in xylen till the coverslip can easily be lifted. Go back through xylen, ethanols till deionisized water. Don't let the section dry out. Then start with antigen-retrieval and so on for the IHC (e.g. DAB and nuclear staining with hematoxylin).
The eosin will be solved by antigen-retrieval. Remaining hematoxylin does not interfere with IHC.
Remove the coverslip putting the slide in xylen or in a xilen-solution, After another passage in xylen, ethanols till deionisized water. But the most important issue is: do not let the section dry out!!! Then you can start the real IHC process! Good luck!
I want to express my sincere gratitude for the answers. A million thanks to you all :)
I would suggest starting with IHC. If we are talking bright-field microscopy any residual background staining from histology may make it difficult to asses IHC results and if you mean IF-IHC there's a lower risk of damaging the tissue when removing a coverslip after IF-IHC than after using DPX or any other mountant that solidifies.
I would suggest that you start with IHC first and then counterstain with haematoxylin after doing the IHC, lightly if you expression is in the cytoplasm . If the protein is expressed in the nucleus then you will stain only the cytoplasm with Eosin and that too very lightly. If your expression is in the nucleus and you counterstain with haematoxylin it will obscure your IHC staining.
Yes.. but I agree with some of the answer above, where hyou are best to begin with I.H.C, then perform a simple stain, like toluidine blue, or haematoxylin and/or Eosin. Use DAB (or like) precipitation stain, NOT I.F, as it will be difficult to get nice images without photo bleaching your I.F.
You can perform the other way around, if you have already stained your tissue with IHC/DAB (not IF).. as said above, depending on you mounting media, soak off the cover slip (xylene if using a hard solvent based mount, PBS/water if using a wet mount) . If a xykene based mount was used follow all the rehydration (ethanol steps) until you get to a saline/PBS step, then perform a IHC stain; use a 10% serum and/or milk powder pre-block step.. this will be essential. The problem by doing post stain IHC is your mount media may have ruined any antigenicity.. and may also provide a super-high background staining. Be very careful never to let your tissue dry out...
Alternatively, if you still have the tissue sections, perform another slice (if possible, a serial one) and stain that; then use deduction that the same region has the same cells; serial sectioning; it's not ideal at all, but may be an otion this late in the game.
good luck!
Can we go back from Xylene to DAB? I mean to say I used my slides for staining a particular Ab and visualizing it with DAB, then counter stained with Cresyl violet. Now they are in Xylene. I can see my specific anbtibody staining but its way too light. So can I restain it with DAB ?
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