I would like to stain a tissue for different markers, and as I there are not enough colours that I can use, I would like to do it sequentially. Thank you in advance!
Hello Belin.
You meet a problem here. Using IHC, you can strip antibodies, but most strong substrates are precipitating. You can use substrates you can strip in alcohol like AEC.
However if you want multicolour IHC and only strip antibodies, just perform HIER (heat epitope retrieval). I use presure cooker (decloacking Chamber) for 5 minutes at 95°C slides in a retrieval buffer (0.05% citraconic anhydrid pH 7.4) as stripping. You can do several rounds With substrates without stripping them, using multispectral camera, seperating the colours after the staining. Look at PerkinElmers solution Vectra.
Olav
Belin,
In Array tomography protocols you can label with one Aby take pics then elute off the Aby and re-label the same section with another Aby:
Elution of Aby’s for c/s re-labeling:
After c/s have been removed with D-H2O, apply elution solution for 20 min rt. (add gently to c/s). Elution solution= 0.2M NaOH, 0.1% SDS in DH2O. (stable at rt for 6 months).
Wash well with 1X TBS 10-15 min.
Wash briefly with D-H2O. aspirate off water.
After sections have dried, place on 65C slide warmer for 30 min.
Store or perform new immunolabeling experiments.
Maybe this can be applied to your needs?
Good Luck
Hi Belin,
My lab experimented with the Array Tomograpgy approach for stripping and reprobing on cultured cells mentioned above by Dr Delannoy. (We tested it out using antibodies against Rab proteins found on different vesicles). It worked, but we had to make a minor modification. The original protocol was applied to plastic sections of brain tissue which can withstand the heating and drying between staining cycles, but cells cannot. So at the end of the stripping procedure, keep the coverslips in PBS until you are ready to start the next staining cycle. We tried this on paraffin sections of brain as well, but we ran into problems of increased autofluorescence from myelin (probably due to long exposure to detergents in the buffers) which created problems. I suspect that stripping probably can be made to work reasonably on tissue sections, but some additional work to adjust the procedure will be needed.
(see Micheva et al., 2010, Cold Spring Harbor Protocols series for a really nice set of papers on the array tomograph approach to analysing multiple antigens in a specimen).
Good Luck.
Dave Sherry
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