Protocol for chemical labeling of Drosophila brains
non cell-permeable substrates (recommended)
- dissect brains in ice-cold 0.1 M PB
- fixation in 4% PFA (in 0.1 M PB) at room temperature for 20 min in glass-well plate on nutator
- SNAP-tag / CLIP-tag: no change in labeling intensity after prolonged fixation of up to 60 min. However, we observed improved labeling intensity for BG-649 substrate after 60 min fixation.
- Halo-tag: labeling intensity significantly decreased with longer fixation (40-60 min)
- TMP-tag: labeling intensity significantly increased with longer fixation (40-60 min)
- transfer brains to 1.5 ml tube, permeabilize briefly (5-10 min) by incubation in 0.5 ml of PBT (0.3% Triton X-100) on rotating wheel
- add substrate for a final concentration of 1 µM in PBT (see Notes on substrates for chemical labeling) higher concentrations can result in higher background
- incubate brains on rotating wheel for 15 min agitation of the sample(s) is important to obtain rapid, homogenous staining! long incubation times (> 6 h) can result in increased background staining!
- wash brains with PBT (2 x 10 min)
- remove PBT as completely as possible and add ~200 µl of Vectashield (or other) mounting medium we observed that subsequently transferring brains into a fresh 200 ~ul aliquot of Vectashield results in more homogenous signal along z
- mount brains on charged slides and image
cell-permeable substrates
- dissect brains in ice-cold 0.1 M PB
- fixation in 4% PFA (in 0.1 M PB) at room temperature for 20 min in glass-well plate on nutator
- SNAP-tag / CLIP-tag: no change in labeling intensity after prolonged fixation of up to 60 min. However, we observed improved labeling intensity for BG-649 substrate after 60 min fixation.
- Halo-tag: labeling intensity significantly decreased with longer fixation (40-60 min)
- TMP-tag: labeling intensity significantly increased with longer fixation (40-60 min)
- transfer brains to 1.5 ml tube, wash briefly (5-10 min) in PBS
- add substrate for a final concentration of 1–5 µM in PBS higher concentrations usually result in considerable background; using cell-permeable substrates in presence of Triton typically results in very weak labeling (when using cell-permeable substrates on permeabilized samples, wash sample with PBS before adding substrate
- incubate brains on rotating wheel for 15 min agitation of the sample(s) is important to obtain rapid, homogenous staining! long incubation times (> 6 h) can result in slightly increased background staining!
- wash brains with PBS, or - to reduce background - with PBT
- remove PBT as completely as possible and add ~200 µl of Vectashield (or other) mounting medium we observed that subsequently transferring brains into a fresh 200 ~ul aliquot of Vectashield results in more homogenous signal along z
- mount brains on charged slides and image
