=====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 [[si:kohl2014:tagging2014substrates|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

=====Recommended fly stocks=====