=====Notes on substrates for chemical labeling=====

     * store substrates at -20°C in presence of dessicant this avoids hydrolysis of substrates 

     * for substrates that are purchased in dry powder form: dissolve in fresh, water-free DMSO using fresh DMSO from a sealed container is crucial as DMSO is hygroscopic; we observed reduced labeling efficiency when using substrates that were dissolved in old DMSO, presumably due to substrate hydrolysis

     * avoid freeze-thawing, aliquot samples (e.g. 2 µl aliquots in 0.2 ml PCR tubes) we observed a decrease in labeling efficiency for some substrates that underwent several freeze-thaw cycles (e.g. BG-649)

     * SNAP Surface488, SNAP Surface549, SNAP Surface649, CLIP Surface547, CLIP Surface488, HaloTag TMR Ligand and TMP Fluorescein work well using our standard staining protocol (see [[si:kohl2014:tagging2014flystain|Protocol for chemical labeling of Drosophila brains]] and [[si:kohl2014:tagging2014mousestain|Protocol for chemical labeling of mouse brain slices]])
 
     * when using chemical labeling in combination with immunostainings, substrates should be added right after tissue permeabilization (not after the o/n blocking step) to obtain optimal results

     * Cost of chemical labeling: the prize of 50 nmol of SNAP substrate (sufficient for ~1,000 labeling reactions at 1 µM concentration in a volume of 0.5 ml each) is comparable to that of an 0.5 ml aliquot of fluorophore-coupled secondary antibody (sufficient for ~1,000 immunostainings in a volume of 0.5 ml each). However, we have successfully tested some substrates (e.g. BG-549, Halo-TMR) at 100 nM and lower concentrations (indeed, there might be situations where lower substrate concentrations - combined with longer incubation times - might be desirable). Also, no primary antibodies and blocking reagents are required for chemical labeling. Therefore, the cost of chemical labeling is comparable to, or potentially lower than, the cost of immunostainings. 

=====Recommended substrates=====
+++ (very good), ++ (decent), + (weak), - (doesn't work)
==SNAP== 
see full list of commercially available substrates [[https://www.neb.com/products/cellular-analysis/snap-tag-substrates|here]]
  * BG-549 (+++)
  * BG-488 (++)
  * BG-649  (++) //longer fixation (40-60 min) improves signal// 
==CLIP==
see full list of commercially available substrates [[https://www.neb.com/products/cellular-analysis/clip-tag-substrates|here]]
  * BC-547 (+++)
  * BC-488 (++)
  * BC-647 (+)
==Halo==
see full list of commercially available substrates [[http://www.promega.co.uk/products/protein-expression-and-mass-spectrometry/protein-labeling-and-detection/halotag-technology-products/halotag-fluorescent-ligands/|here]]
  * TMR (+++) expensive - use at low concentration (< 1:2,000)
==TMP==
see full list of commercially available substrates [[http://www.activemotif.com/catalog/122/ligandlink-universal-protein-labeling|here]]
  * Fluorescein v2 (from Virginia Cornish)