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Tissue staining with chemical tags

For protein localization studies, enzymes can be fused to proteins in the same way as fluorescent protein tags such as GFP. Tissues can be fixed and exposed to chemical substrates that covalently modify the enzyme tags with fluorescent molecules. This approach avoids problems often associated with detecting fluorescent protein tags with antibodies while providing a fast detection method.

The figure shows the use of four commercially available enzyme-substrate systems: SNAP, CLIP, TMP and Halo.

Figure of chemical tags originating in Kohl et al. (2014), Ultrafast tissue staining with chemical tags. PNAS 111:E3805-E3814.

Methods are described in Kohl et al. (2014). Ultrafast tissue staining with chemical tags. PNAS 111: E3805–E3814.

Newer versions of several of these constructs are now available. For more information, see Sutcliffe et al. (2017). Second Generation Drosophila Chemical Tags: Sensitivity, Versatility and Speed. Genetics 205(4): 1399–1408.

The following table shows stocks for expressing enzyme-tagged proteins that can be detected with these chemical substrates.

  Stock can be used to label: Tag Stk # Genotype
1 actin (LifeAct) Halo 67625 w[*]; P{y[+t7.7] w[+mC]=UAS-LifeAct::Halo2}attP40/CyO
2 cell membranes CLIP 67612 w[*]; Kr[If-1]/CyO; PBac{y[+mDint2] w[+mC]=LexAop2-myr::4xCLIPf}VK00005
3 cell membranes CLIP 67610 w[*]; Kr[If-1]/CyO; PBac{y[+mDint2] w[+mC]=UAS-myr::4xCLIPf}VK00005
4 cell membranes CLIP 67613 w[*]; P{y[+t7.7] w[+mC]=LexAop2-myr::4xCLIPf}attP40; MKRS/TM6B, Tb[1]
5 cell membranes CLIP 58392 w[*]; P{w[+mC]=UAS-CD4-CLIPf}2/CyO; Ki[1]/TM6B, Tb[1]
6 cell membranes CLIP 58390 w[*]; P{w[+mC]=UAS-CD4-CLIPf}3
7 cell membranes CLIP 67611 w[*]; P{y[+t7.7] w[+mC]=UAS-myr::4xCLIPf}attP40/CyO; MKRS/TM6B, Tb[1]
8 cell membranes Halo 67620 w[*]; P{y[+t7.7] w[+mC]=UAS-3xHalo7::CAAX}attP40
9 cell membranes Halo 67621 w[*]; P{y[+t7.7] w[+mC]=UAS-7xHalo7::CAAX}attP40
10 cell membranes Halo 67619 w[*]; P{y[+t7.7] w[+mC]=UAS-Halo7::CAAX}attP40
11 cell membranes Halo 58395 w[*]; Kr[If-1]/CyO; P{y[+t7.7] w[+mC]=UAS-myr-Halo}attP2
12 cell membranes Halo 58394 w[*]; P{y[+t7.7] w[+mC]=UAS-myr-Halo}attP40
13 cell membranes Halo 58396 w[*]; P{y[+t7.7] w[+mC]=UAS-myr-Halo}attP40/CyO; P{y[+t7.7] w[+mC]=UAS-myr-Halo}attP2
14 cell membranes SNAP 67618 w[*]; PBac{y[+mDint2] w[+mC]=LexAop2-myr(Bxb.HA-STOP)4xSNAPf}VK00018; MKRS/TM6B, Tb[1]
15 cell membranes SNAP 67632 w[*]; PBac{y[+mDint2] w[+mC]=LexAop2-myr(Bxb.HA-STOP)4xSNAPf}VK00018; PBac{y[+mDint2] w[+mC]=UAS(-FRT)Bxb1}VK00027
16 cell membranes SNAP 67633 w[*]; P{y[+t7.7] w[+mC]=GMR57C10-lexA}attP40 PBac{y[+mDint2] w[+mC]=LexAop2-myr(Bxb.HA-STOP)4xSNAPf}VK00018/CyO; PBac{y[+mDint2] w[+mC]=UAS(-FRT)Bxb1}VK00027
17 cell membranes SNAP 67616 w[*]; PBac{y[+mDint2] w[+mC]=LexAop2-myr::4xSNAPf}VK00005/TM6B, Tb[1]
18 cell membranes SNAP 67617 w[*]; Kr[If-1]/CyO; PBac{y[+mDint2] w[+mC]=UAS-myr(Bxb.HA-STOP)4xSNAPf}VK00005/MKRS
19 cell membranes SNAP 67614 w[*]; Kr[If-1]/CyO; PBac{y[+mDint2] w[+mC]=UAS-myr::4xSNAPf}VK00005/TM6B, Tb[1]
20 cell membranes SNAP 67652 w[*]; P{y[+t7.7] w[+mC]=LexAop2-myr::4xSNAPf}attP40; MKRS/TM6B, Tb[1]
21 cell membranes SNAP 58389 w[*]; P{w[+mC]=UAS-CD4-SNAPf}2/CyO
22 cell membranes SNAP 58388 w[*]; P{w[+mC]=UAS-CD4-SNAPf}3/TM3, Sb[1]
23 cell membranes SNAP 58375 w[*]; Kr[If-1]/CyO; P{y[+t7.7] w[+mC]=UAS-myr-SNAPf}attP2/TM6B, Tb[1]
24 cell membranes SNAP 58376 w[*]; P{y[+t7.7] w[+mC]=UAS-myr-SNAPf}attP40/CyO; Ki[1]/TM6B, Tb[1]
25 cell membranes SNAP 67615 w[*]; P{y[+t7.7] w[+mC]=UAS-myr::4xSNAPf}attP40; MKRS/TM6B, Tb[1]
26 cell membranes TMP 58391 w[*]; P{y[+t7.7] w[+mC]=UAS-myr-TMP}attP40/CyO; P{y[+t7.7] w[+mC]=UAS-myr-TMP}attP2
27 cell membranes and pre-synaptic T-bars Halo, SNAP 58399 w[*]; brp[T:ZzzzSNAP-f], P{w[+mC]=alphaTub84B(FRT.GAL80)}2/CyO; P{y[+t7.7] w[+mC]=UAS-myr-Halo}attP2
28 cell membranes, synaptic vesicles and pre-synaptic T-bars Halo, CLIP, SNAP 58398 w[*]; brp[T:ZzzzSNAP-f], P{w[+mC]=UAS-CLIPf-nSyb}2/CyO; P{y[+t7.7] w[+mC]=UAS-myr-Halo}attP2
29 ChemBrainbow Halo, CLIP, SNAP 67630 w[*]; Kr[If-1]/CyO; PBac{y[+mDint2] w[+mC]=UAS-ChemBrainbow.SCH}VK00005/TM6B, Tb[1]
30 ChemBrainbow Halo, CLIP, SNAP 67631 w[*]; Kr[If-1]/CyO; PBac{y[+mDint2] w[+mC]=UAS-ChemBrainbow.SCH}VK00005 P{y[+t7.7] w[+mC]=UAS-ChemBrainbow.SCH}attP2/TM6B, Tb[1]
31 ChemBrainbow Halo, CLIP, SNAP 67629 w[*]; Kr[If-1]/CyO; P{y[+t7.7] w[+mC]=UAS-ChemBrainbow.SCH}attP2
32 ChemBrainbow Halo, CLIP, SNAP 67631 w[*]; Kr[If-1]/CyO; PBac{y[+mDint2] w[+mC]=UAS-ChemBrainbow.SCH}VK00005 P{y[+t7.7] w[+mC]=UAS-ChemBrainbow.SCH}attP2/TM6B, Tb[1]
33 dendrites and neuronal cell bodies CLIP 58381 w[*]; P{w[+mC]=UAS-TLN-CLIPm}2/CyO
34 dendrites and neuronal cell bodies CLIP 58382 w[*]; P{w[+mC]=UAS-TLN-CLIPm}3
35 dendrites and neuronal cell bodies SNAP 58384 w[*]; P{w[+mC]=UAS-TLN-SNAPm}2/CyO
36 dendrites and neuronal cell bodies SNAP 58383 w[*]; P{w[+mC]=UAS-TLN-SNAPm}3/TM3, Sb[1]
37 pre-synaptic T-bars SNAP 58397 w[*]; brp[T:ZzzzSNAP-f]/CyO; Ki[1]/TM6B, Tb[1]
38 synaptic vesicles (nSyb) CLIP 58393 w[*]; P{w[+mC]=UAS-CLIPf-nSyb}2/CyO; Ki[1]/TM6B, Tb[1]
39 synaptic vesicles (nSyb) CLIP 58385 w[*]; P{w[+mC]=UAS-CLIPf-nSyb}3/TM3, Sb[1]
40 synaptic vesicles (nSyb) SNAP 58386 w[*]; P{w[+mC]=UAS-SNAPf-nSyb}2/CyO
41 synaptic vesicles (nSyb) SNAP 58387 w[*]; P{w[+mC]=UAS-SNAPf-nSyb}3/TM3, Sb[1]
42 synaptic vesicles (Syt1) CLIP 58378 w[*]; P{w[+mC]=UAS-Syt1-CLIPm}2
43 synaptic vesicles (Syt1) CLIP 58377 w[*]; P{w[+mC]=UAS-Syt1-CLIPm}3/TM3, Sb[1]
44 synaptic vesicles (Syt1) Halo 67623 w[*]; P{y[+t7.7] w[+mC]=UAS-Syt1::3xHalo7}attP40
45 synaptic vesicles (Syt1) Halo 67624 w[*]; P{y[+t7.7] w[+mC]=UAS-Syt1::7xHalo7}attP40
46 synaptic vesicles (Syt1) Halo 67622 w[*]; P{y[+t7.7] w[+mC]=UAS-Syt1::Halo7}attP40
47 synaptic vesicles (Syt1) SNAP 58380 w[*]; P{w[+mC]=UAS-Syt1-SNAPm}2/CyO
48 synaptic vesicles (Syt1) SNAP 58379 w[*]; P{w[+mC]=UAS-Syt1-SNAPm}3/TM3, Sb[1]