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
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