Preparing tissue sample for dSTORM imaging is not that different than preparing them for conventional microscopy.
Recommendation:
- Your sample can either be cryosection or Paraffin Embedded Tissues - FFPE.
- If you have the choice we would recommend to do cryosection as the whole sample preparation is easier that way.
- The most important part is to put your tissue on a 1.5 coverslip and not on the slide!
- Having thinner slice (less than 10um) is better but the only disadvantage of thicker slice is that you will only be able to image the bottom part of the tissue.
- It may be good to add a membrane marker to navigate in the tissue and visualise cell type of interest
- Remember to prepare some controls
- Put fresh tissue in OCT freezing media and snap froze
- Coat 1.5 coverslips with Poly-l-lysine (PLL) or 0.03% gelatin
- Use a cryostat to slice sections (fatty tissues can be hard to cut thin)
- Put the sliced sections on the coated coverslips
- Rehydrate tissues with water and gently wash OCT media with PBS
- Fix tissues for 30 min using 4% PFA or PFA+GLU mix (4% and 0.2%)
- Quench fixation with Glycine (25mM) for 10 min
- Permeabilize tissues (if needed) with 0.1–0.5% Tween-20 and 5% bovine serum albumin [BSA] in PBS
- Incubate your tissues with antibodies (1h at 2 μg/ml is a good starting point)
- After wash, perform a post fixation as previously explained
- Perform quenching as previously explained
- Add dSTORM buffer and image under the microscope
Protocol for FFPE:
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