Dear Researchers,
I have a batch of slides (mouse muscle cryosections) from a pretty precious origin, and I try to make the best use of every slide.
I made a staining with an antibody (Alexa 488) and with a-bungarotoxin (Alexa 594 conjugate). Anyway, antibody staining looked rather unspecific, so I had meaningful signals only from the 594 channel (and DAPI).
I am curious if I can do classic H&E staining with my slides, simultaneously retaining the a-bungarotoxin signal on the 594 channel?
Best,
Michail
Dear Machail
H& E staining might interfere with your Antibodies , so
You can performed it in sequential manner.
Good Luck !!
Hi Michail,
I did this experiment before, I started with the IF (read my slides) and after I performed HE stainings and it worked very well.
Good luck
I have contrasted triple-stained IF slides with hematoxylin without any trouble using Shandon Instant Hematoxylin #6765015. Without a free channel for nuclear stain but need for orientation, this solution worked well, although not easy to grab pictures. But it might mask a nuclear signal if you stain for that.
Eosin is a dye with fluorescence over a broad wavelength and will pass many filter cubes.
I would not recommend eosin when using DAB as proposed of others unless you turn it into black with nickel enhancement, since it can be hard to discriminate weakly brown staining from orange unless you use spectral imaging (PerkinElmer Vectra/Mantra).
We bought a Cy5 filter cube catch 4 signals on an ordinary fluorescence microscope (Nikon i80 with DS-Ri1 camera): DAPI, Cy2, Cy3 and Cy5. You can't see Cy5, but so does the camera!
Good luck!
In fact I didn't yet try to combine the HE staining with immunofluorescence, as we started with the immuno only now. In any case I would like to try to combine the two techniques. It will necessitate some thinking in advance and experiments concerning a selection of chemicals, steps of staining and of kind of mounting substance.
Actually I just checked my HE slides for fluorescence and they were brightly shining in all channels. So probably the best strategy is to do Immuno first and record the position of the image, and repeat the imaging in the BF after the HE staining at the same XY coordinates.
Any protocol we can use for h and e? We find that eosin stains but hematoxylin is not staining the nuclei.
We did did a dual IF stain followed by the H&E.
Always start sections from aqueous state. For FFPE deparaffinise and hydrate through graded ethanol 100-100-96-96-70% to water. I use Shandon instant hematoxylin as it is easy to prepare (www.thermofisher.com/order/catalog/product/6765015), 1 minute for H&E, much shorter for counterstain after immunostaining (5-10 sec in a 50% dilution of hematoxylin). Wash thoroughly with running tap water for 5-10 min. Bluing can be done by 2 min in PBS for the weakly stained immuno-slides, or for H&E, 2-5 min in ammonium-water or 5% w/v hexamethylenetetramine in water. Wash again before staining with 0.5 % w/v eosin-Y (1B 425; addition of 0.2% acetic acid will even intensify eosin) in water for 3 min. Wash and dehydrate quickly (eosin washes out in 70 and 96% etOH) in graded ethanol 70-96-96-100-100% before xylene and mounting.
Once again: do NOT stain with eosin when using fluorescence or brown DAB substrate if you do not have a spectral imaging system for unmixing, if possible at all! Eosin is highly fluoresce, while hematoxylin is not.
AND: not all fluorophores and substrates can be dehydrated.
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