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The above notes contain full details for sample preparation but here are some key points to remember about STED sample preparation and imaging for fixed specimens. Please read them so you don't make any major mistakes in sample prep:
- Use commercially prepared formaldehyde that is free from formic acid and sealed in vials. There have been reports of high background using old formaldehyde that had been prepared from paraformaldehyde. I recommend Pierce™ 16% Formaldehyde (w/v), Methanol-free (Thermo 28906)
- You must not have DAPI or Hoechst in your specimen because they cause high background with the 592 nm depletion laser.
- We only have a 592 nm depletion laser, so all dyes must emit in the green region of the spectrum. Please see the note below about dyes for one-colour and two-colour STED
- Use antibodies at higher concentrations than normal. Leica recommends using most commercially available antibodies at a 1:100 dilution.
- Perform several rinses after each antibody incubation and then wash at least three times for extended periods of 10 to 20 minutes each
- Do not use Vectashield as a mountant as it reduces the brightness of long Stokes shift dyes
- The recommended mountants are: Prolong Diamond without DAPI, Mowiol (see Leica's recipe in the tech notes above), Prolong Gold without DAPI
- The observed structure should preferably be no more than 20 µm from the cover glass.
- If you have a red or far red counter stain (e.g. Alexa 568, Alexa 647) then you must image that channel before capturing a STED channel because the 592 nm depletion laser will bleach the red dye.
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