We normally do free-floating sections for DAB-based IHC- which means at the end, the sections are mounted onto slides, air dried, and then coverslipped.
When doing fluorescent IHC on free-floating sections, should we also mount, air dry, and then coverslip or just mount and carefully coverslip when it is wet?
I wash the sections with a final rinse of Tris buffer to remove salt from PBS washes, mount sections onto gelatin subbed slides and then air-dry sections lightly, you want them to adhere to the slide but not dry out completely. If they are very wet they will not stick to the slide and move under the coverslip. Coverslip with Prolong Gold and allow to dry overnight. Store the slides in the refrigerator.
I mount them with a brush with a bit of PBS and I let them dry. Indeed, if it's still wet you will have problems when you add the coverslip : sections can fold and/or move, especially if they are thin. But on the other hand, you don't want of course to let them dry too much, or you will lose your fluorescence and even damage the tissue.
After drying, I coverslip using Vectashild with/without DAPI, seal with nail polish and store at -20°C.
We typically mount and air dry (but not the overnight drying that we did before clearing and coverslipping our DAB labeled sections). We typically let the sections dry for about 15min to a half hour (til they are visibly drier but not bone dry or we find we get a lot of bubbles) then use vectashield hardset mounting medium (with dapi) and let those dry overnight to let the medium set.
Some other things we changed when transitioning from DAB to fluorescence were:
1) increased the permiablization (we use 1.5% triton-x in our blocking buffer and incubate for 2hrs for fluorescence).
2) increase the concentration of the primary and secondary antibodies (we usually use our primary antibodies at 1:200-1:1000 and our secondaries at 1:250-1:500 depending on the antibody but it is always more concentrated than when using DAB).
3) We don't clear the slides by running then through alcohols after staining when doing fluorescence (which we always do when labeling with DAB).
4) we don't use permount mounting medium because that will quench your fluorescent signal. We have used the vectashlied (either hardset or regular will work), prolong gold (warm slowly without shaking to reduce the bubbles), and DPX (which works very much like permount without the quenching but is very toxic). If it is a non-hardset mounting medium we use clear nail polish to seal the coverslips.
Hope this helps.
I agree with Audrey, but I would add that the Vectashield and Prolong Gold mounting medias work best when the tissue is still somewhat wet. I suggest you apply some of the mounting media to the slide and unroll the free float section into the mounting media straight from PBS, then add more mountant on top and coverslip. Avoid bubbles at all steps! Dry the sample flat overnight before imaging, and seal the edges with clear nailpolish to maintain the sample. (Prolong changes refractive index overnight and images will improve with curing). You also need to store fluorescent samples in the dark at 4C or -20 to maintain fluorescent signals.
For DPX, you would dehydrate and run through solvents Xylene or Toluene before mounting-it behaves like Permount but doesn't give autofluorescence.
For Immuno-fluorescence, it is necessary to mount the slides in aqueous phase. It is better to use a specific mounting medium for fluorescence to avoid fading. For example, "Fluorescent mounting medium" (Dako), "Fluoromount G" (Southern Biotech) or "FluorSave Reagent" (Calbiochem). For my part, I have slides mounted with Dako medium in the refrigerator for more than 4 years that are perfectly usable.
Yes, you have to mount, air dry at dark and coverslip when it is totally dry also at very little light.
We recommend always keeping the tissues wet and not completely drying when doing fluorescence work-they mount more easily and you get less background problems.
hold on a second. many folks above said mounting the slices when they are wet-ish. but in that way you had problem with non-homogeneous tissue with some water some mounting medium. You won't be able to imaging very deep under confocal due to aberration. am I missing something or people just image the first 20 um only?
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