Figures and data in Two-step regulation of trachealess ensures tight coupling of cell fate with morphogenesis in the Drosophila trachea

Figures
Tables
Additional files

5 figures, 1 table and 2 additional files

Figures

Figure 1 with 3 supplements

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*trh* is essential for maintaining the invaginated tracheal structures.

(A) Schematic of the tracheal morphogenesis process. For clarity, only apical surfaces are shown. (**B, C**) Live imaging of tracheal invagination in a control embryo (B) and a *trh* mutant (C). Red arrowheads: apical constriction forming a tracheal pit. Yellow circle: a mitotic cell associated with accelerated invagination, distinguished by condensed histone. Red arrows: transient invagination and return to epidermis in a *trh¹* mutant. Gray arrows: segmental groove, which is not a tracheal structure. Par-6::GFP indicates the apical cell side, and His2Av::mRFP indicates chromosomes. Time point zero is set to the onset of germband retraction. (D) Activity of *R14E10-GAL4* in a control embryo monitored using *UAS-mCD8RFP*. (**E–G**) Activity of *R14E10-GAL4* in *trh^A14-3/B16-11* mutant embryos monitored using *UAS-mCD8RFP*. Green: DE-cad, Magenta: mCD8RFP driven by *R14E10-GAL4*. Cells expressing RFP initiated invagination at stage 10 (E), and invaginated structures formed within the RFP-positive cell cluster at stage 12 (F). However, these invaginated structures were not observed at stage 16, and RFP-positive cells were observed in the surface epidermis (G). Scale bars, 10 μm.

https://doi.org/10.7554/eLife.45145.003

Figure 1—figure supplement 1

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Activation pattern of EGFR signaling before invagination.

Activation pattern of ERK (di-phosphorylated ERK) in a control, a *trh^A14-3* mutant, and a *rho^del1* mutant embryo at stage 10 before invagination. Before invagination, ERK was phosphorylated in the tracheal placodes of the control and *trh* mutant but not in those of the *rho* mutant. tracheal placodes were labeled by using *trh66-lacZ*.

https://doi.org/10.7554/eLife.45145.004

Figure 1—figure supplement 2

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Characterization of TALEN-induced *trh* mutants.

(A) Predicted amino acid sequences of *trh^A14-3* and *trh^B16-11* TALEN mutant proteins. Each allele has a frameshift mutation on the most upstream common exon among all isoforms. Both frameshift mutations lead to premature translational termination and the loss of the PAS A, PAS B and transcription activation domains. The *trh^A14-3* allele also loses the bHLH domain. The changes in the predicted amino acid sequences of the Trh-PA isoform are shown as an example. (B) No Trh protein expression was detected in *trh^A14-3/B16-11* mutant embryos. β-gal driven by *R14E10-GAL4* labels would-be tracheal cells.

https://doi.org/10.7554/eLife.45145.005

Figure 1—figure supplement 3

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The *trh* mutant phenotype was not rescued by *btl* overexpression or the inhibition of apoptosis.

(**A–D**) Phenotypes resulting from gene overexpression in the tracheal placodes of *trh^A14-3/B16-11* mutants. Each gene was overexpressed using *R14E10-GAL4*. (A) *trh* overexpression, (B) *btl:GFP* overexpression, (C) *p35* overexpression. Scale bars, 10 μm.

https://doi.org/10.7554/eLife.45145.006

Figure 2 with 1 supplement

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Trh expression is maintained only in invaginated tracheal cells.

(**A, B**) Trh expression in a tracheal placode of a control (*rho^del1 bnl^P1* /+) (a) and a *rho^del1 bnl^P1* mutant (B) embryo at stage 10 before invagination. (**C–E**) Trh and β-gal expression in a control embryo (C), a *rho^del1 bnl^P1* mutant (D), and a *rho^del1 bnl^P1* mutant with p35 overexpression (E) at stage 14 after invagination. β-gal and p35 expression were driven by *R14E10-GAL4*. (F) Schematic of the tracheal branching pattern after invagination. (G) Upper: Boxplot of cell numbers. Initial Trh+: the number of Trh-expressing cells before invagination, *trh*-Off: the number of cells expressing β-gal driven by *R14E10-GAL4* in the epidermis (Trh-negative) after invagination (stage 13–14), *trh*-On: the number of invaginated tracheal cells expressing Trh after invagination (stage 13–14), sum: the sum of *trh*-Off and *trh*-On. **: Exact Wilcoxon-Mann-Whitney Test, p=0.001331, *: Steel-Dwass test, p<0.001 (for *trh*-Off, control vs *rho bnl: p*=5.3 × 10⁻⁹, control vs *rho bnl +p35: p*=8.3 × 10⁻¹⁰, *rho bnl* vs *rho bnl +p35: p*=3.9 × 10⁻⁸; for *trh*-On, control vs *rho bnl: p*=5.28 × 10⁻¹⁰, control vs *rho bnl +p35: p*=8.4 × 10⁻¹⁰, *rho bnl* vs *rho bnl +p35: p*=2.8 × 10⁻¹⁰; for sum, control vs *rho bnl: p*=3.1 × 10⁻¹⁰, control vs *rho bnl +p35: p*=2.0 × 10⁻⁴, *rho bnl* vs *rho bnl +p35: p*=3.2 × 10⁻⁹). Lower: mean numbers of *trh*-On cells and *trh*-Off cells at stages 13–14 after invagination. (H) Schematic of the dynamics of Trh expression during invagination. Scale bars, 10 μm.

https://doi.org/10.7554/eLife.45145.007

Figure 2—source data 1 Source data for Figure 2G and Figure 2—figure supplement 1B.: https://doi.org/10.7554/eLife.45145.009
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Figure 2—figure supplement 1

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Only a few invaginated cells maintain Trh expression in *rho CycA bnl* mutants.

(A) Trh expression in the tracheal cells of a *rho^del1 CycA^C8LR1 bnl^P1* mutant at stage 10 before invagination and stage 14 after invagination. Although Trh expression was almost normal at stage 10, only a few invaginated cells were Trh-positive after invagination. Scale bars, (stage 10) 50 μm, (stage 14) 10 µm. (B) Boxplot showing the numbers of Trh+ cells in each placode (tr4, 5 and 6) at stage 10 before invagination and stage 14 after invagination.

https://doi.org/10.7554/eLife.45145.008

Figure 3 with 3 supplements

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*R15F10* reproduces the tubule-restricted Trh pattern.

(A) Genomic positions of *R15F01*, other enhancers, and the insertion site of *1-eve-1* at the *trh* locus (**B–D**) Enhancer activity of *R15F01* monitored using *R15F01-GAL4* with *UAS-mCD8RFP* in a control (B’), *rho^del1 bnl^P1* mutant (C), and *rho^del1 CycA^C8LR1 btl^deltaOh10* mutant (D) embryo. β-gal indicates the expression from a lacZ enhancer trap line for *trh*, *1-eve-1* (B). (E) Phenotype in adult cuticle pigmentation. The *R15F01-GAL4* fly had a lighter body color than the *R14E10-GAL4* fly, indicating that *R15F01* represses an adjacent *mini-yellow* gene. (**F–M**) Enhancer activity of *R15F01* monitored using the direct *lacZ* reporter in control embryos and in several invagination mutants: *rho^del1 CycA^C8LR1* (H), *rho^del1 bnl^P1* (I), *CycA^C8LR1 bnl^P1* (J), *rho^del1 CycA^C8LR1 bnl^P1* (K), *Df(1)BSC352* (deficient in all *upd1, 2* and 3), arrowhead: a segment with invaginated trachea, arrow: a segment without trachea (L), and *trh^A14-3/B16-11 R14E10-GAL4* > *UAS-flag::trh[S665D]* (M). Scale bars, 10 μm.

https://doi.org/10.7554/eLife.45145.010

Figure 3—figure supplement 1

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Activity of *trh* enhancers in tracheal cells and surrounding epidermal cells.

(A) The *trh47* activity in control and *rho^del1 bnl^P1* mutant embryos. In both cases, cells activating the *trh47* element were observed in both the trachea and epidermis at stage 14 after invagination. Scale bars, 10 μm. (B) The *trh67* activity in control and *rho^del1 bnl^P1* mutant embryos. In both cases, the *trh67* element was activated in tracheal cells in a sporadic manner. Scale bars, 10 μm.

https://doi.org/10.7554/eLife.45145.011

Figure 3—figure supplement 2

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Activity of *R14E10* and *R15F01* in *rho bnl* and *trh* mutants.

(A) Schematic of the GAL4- or lacZ transgene structure after integration into the *attP2* or *attP40* site. (B) β-gal reporter expression in a *R14E10-lacZ* strain at stage 10 (before invagination). (C) Boxplot of the number of β-gal positive cells in each placode (tr4, 5 and 6). n = 15. (**D–E**) Enhancer activity of *R15F01* monitored using the direct *lacZ* reporter at stage 10 (before invagination): in a *rho^del1 bnl^P1* mutant (d) and a *trhA^14-3* mutant (e). (F) Enhancer activity of *R15F01* monitored using the direct *lacZ* reporter at stage 15 in a *trhA^14-3* mutant. (G) Boxplot of the number of β-gal positive cells in each placode (tr4, 5 and 6) at stage 10 (before invagination). n = 18 (control), n = 15 (*rho bnl*), n = 15 (*trh*). *: Steel-Dwass test, p<0.01 (control vs rho bnl: p=0.0027, control vs trh: p=5.8 × 10⁻⁶, rho bnl vs trh: p=5.0 × 10⁻⁴. (H) Boxplot of the number of β-gal positive cells in each placode (tr4, 5 and 6) at stage 15 in *trh* mutants. n = 15.

https://doi.org/10.7554/eLife.45145.012

Figure 3—figure supplement 2—source data 1 Source data for Figure 3—figure supplement 2C,G,H.: https://doi.org/10.7554/eLife.45145.013
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Figure 3—figure supplement 3

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*trh*-overexpression by using R15F01-GAL4 rescued the *trh* mutant phenotype in maintaining invaginated structures.

*trh*-OE using *R14E10-GAL4* rescued both tracheal invagination and the maintenance of invaginated structures of *trh* mutants. On the other hand, *trh*-OE using *R15F01-GAL4* did not rescue the invagination phenotype at stage 12, which may be due to the delayed onset of *R15F01* and/or a smaller number of R15F01-positive cells, but allowed cells to keep invaginated structures later.

https://doi.org/10.7554/eLife.45145.014

Figure 4 with 1 supplement

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The *R15F10* CRM contains multiple tracheal enhancers and epidermal silencers.

(A) Summary of the domain mapping of *R15F01*. ± indicates sporadic expression. (**B–E**) Activities of *R15F01* (full length), D1, D7, and del4 monitored using GAL4 and *UAS-mCD8RFP*. D7 activated RFP expression in both tracheal and surrounding epidermal cells (arrows in E). D1 induced RFP expression in a sporadic manner. (**F–I**) Activities of D1 and D7 monitored using the direct *lacZ* reporter at stage 10 (before invagination), stage 11 (during invagination), and stage 15 (after invagination). D7 showed enhancer activity in the tracheal placodes before invagination, but D1 activated the reporter in tracheal cells after invagination. Arrows in I indicate epidermal expression of β-gal. Scale bars, 10 μm.

https://doi.org/10.7554/eLife.45145.015

Figure 4—figure supplement 1

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Enhancer and silencer activities of truncated *R15F01* elements.

(A) Adult body colors of each transgenic strain. (**B–G**) β-gal reporter expression in D2, D3, D4, D5, D6, and D8-lacZ strains at stage 14 or 15 after invagination. (**H–K**) β-gal reporter expression in del1, del2, del3, and del4-lacZ strains at stage 10 (before invagination), stage 11 (during invagination), and stage 14 (after invagination). del1-lacZ did not show β-gal expression before invagination and showed sporadic tracheal expression after invagination. Scale bars, 10 μm.

https://doi.org/10.7554/eLife.45145.016

Figure 5

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The *R15F01* epidermal silencers counteract a heterologous epidermal enhancer.

(A) Schematic of chimeric reporters with *svb-E6B* and *R15F01* fragments (**B, C**) Reporter β-gal expression (magenta or fire) from *E6B-lacZ* and *E6B-[D3-6]-lacZ* integrated at the *attP40* site. The epidermal β-gal expression in *E6B-[D3-6]-lacZ* was significantly weaker than that in *E6B-lacZ*. (**D–F**) Reporter β-gal expression (magenta or fire) from *E6B-lacZ*, *E6B-[D1-2]-lacZ*, and *E6B-[del1]-lacZ* integrated at the *ZH-51c* site. The epidermal β-gal expression in *E6B-[D1-2]-lacZ* and *E6B-[del1]-lacZ* was significantly weaker than that in *E6B-lacZ*, while both showed tracheal reporter expression. (**G–I**) Reporter GFP expression (green or fire) in (G) *arm-GAL4* >*3×UAS-GAP43GFP*, (H) *arm-GAL4* >*3 × UAS-R15F01-GAP43GFP*, and (I) *3×UAS-GAP43GFP* only. Both *3×UAS-GAP43GFP* and *3×UAS-R15F01-GAP43GFP* transgenes were integrated at the *attP2* site. The epidermal GFP expression (the surface of embryos) in *arm-GAL4* >*3×UAS-R15F01-GAP43GFP* was significantly weaker than that of *3×UAS-GAP43GFP*, while hindgut GFP expression was detectable. Embryos possessing only *3×UAS-GAP43GFP* showed tracheal GFP expression but not hindgut expression. Arrows indicate the hindgut.

https://doi.org/10.7554/eLife.45145.017

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (Drosophila melanogaster)	trh[1]	Kyoto stock center	DGRC:106845; FLYB: FBal0017036
Genetic reagent (Drosophila melanogaster)	trh[A14-3]	Kondo et al., 2014	FLYB: FBal0344676
Genetic reagent (Drosophila melanogaster)	trh[B16-11]	Kondo et al., 2014	FLYB: FBal0344695
Genetic reagent (Drosophila melanogaster)	1-eve-1	Bloomington Drosophila Stock Center	BDSC: 8744; FLYB: FBti0002897	FlyBase symbol: P{ET-L}trh-1-eve-1
Genetic reagent (Drosophila melanogaster)	R14E10-GAL4(attP2)	Bloomington Drosophila Stock Center	BDSC: 48641
Genetic reagent (Drosophila melanogaster)	R15F01-GAL4(attP2)	Bloomington Drosophila Stock Center	BDSC: 45071; FLYB: FBti0133347	FlyBase symbol: P{GMR15F01-GAL4}attP2
Genetic reagent (Drosophila melanogaster)	UAS-mCD8.ChRFP	Bloomington Drosophila Stock Center	BDSC: 27392; FLYB: FBti0115769	FlyBase symbol: P{UAS-mCD8.ChRFP}3
Genetic reagent (Drosophila melanogaster)	UAS-nls-lacZ	Kyoto stock center	DGRC:108782; FLYB: FBti0002781	FlyBase symbol: P{UAS-GFP::lacZ.nls}30.1
Genetic reagent (Drosophila melanogaster)	UAS-flag::trh	PMID: 11740943	FLYB: FBal0150204
Genetic reagent (Drosophila melanogaster)	UAS-flag::trh[S665D]	PMID: 11740943	FLYB: FBal0150205
Genetic reagent (Drosophila melanogaster)	UAS-btl::GFP	Bloomington Drosophila Stock Center	BDSC: 41802; FLYB: FBti0148917	FlyBase symbol: P{UAS-btl::GFP-S65T}3
Genetic reagent (Drosophila melanogaster)	UAS-p35	Kyoto stock center	DGRC: 108018; FLYB:FBti0012594	FlyBase symbol: P{UAS-p35.H}BH1
Genetic reagent (Drosophila melanogaster)	rho[del1]	PMID: 2110920	FLYB: FBal0017860
Genetic reagent (Drosophila melanogaster)	bnl[P1]	Bloomington Drosophila Stock Center	BDSC: 6384; FLYB: FBal0057745
Genetic reagent (Drosophila melanogaster)	CycA[C8LR1]	Bloomington Drosophila Stock Center	BDSC: 6627; FLYB: FBal0065308
Genetic reagent (Drosophila melanogaster)	btl[deltaOh10]	Ohshiro and Saigo, 1997	FLYB: FBal0083056
Genetic reagent (Drosophila melanogaster)	Df(1)BSC352	Bloomington Drosophila Stock Center	BDSC: 24376; FLYB: FBab0045128
Genetic reagent (Drosophila melanogaster)	Par6::GFP	PMID: 18854163	FLYB: FBal0243990
Genetic reagent (Drosophila melanogaster)	His2Av::mRFP	Bloomington Drosophila Stock Center	BDSC: 23651; FLYB: FBti0077845	FlyBase symbol: P{His2Av-mRFP1}II.2
Genetic reagent (Drosophila melanogaster)	His2Av::mRFP	Bloomington Drosophila Stock Center	BDSC: 23650; FLYB: FBti0077846	FlyBase symbol: P{His2Av-mRFP1}III.1
Genetic reagent (Drosophila melanogaster)	arm-GAL4[11]	Kyoto stock center	DGRC:106387; FLYB: FBti0002793	FlyBase symbol: P{GAL4-arm.S}11
Genetic reagent (Drosophila melanogaster)	trh66-lacZ	Sotillos et al., 2010	FLYB: FBal0265118
Genetic reagent (Drosophila melanogaster)	R15F01-lacZ(attP40)	this paper	N/A	lacZ reporter with R15F01 fragment
Genetic reagent (Drosophila melanogaster)	R14E10-lacZ(attP40)	this paper	N/A	lacZ reporter with R14E10 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-D1-lacZ(attP40)	this paper	N/A	lacZ reporter with R15F01-D1 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-D2-lacZ(attP40)	this paper	N/A	lacZ reporter with R15F01-D2 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-D3-lacZ(attP40)	this paper	N/A	lacZ reporter with R15F01-D3 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-D4-lacZ(attP40)	this paper	N/A	lacZ reporter with R15F01-D4 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-D5-lacZ(attP40)	this paper	N/A	lacZ reporter with R15F01-D5 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-D6-lacZ(attP40)	this paper	N/A	lacZ reporter with R15F01-D6 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-D7-lacZ(attP40)	this paper	N/A	lacZ reporter with R15F01-D7 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-D8-lacZ(attP40)	this paper	N/A	lacZ reporter with R15F01-D8 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-del1-lacZ(ZH-51C)	this paper	N/A	lacZ reporter with R15F01-del1 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-del1-lacZ[FS](attP40)	this paper	N/A	lacZ reporter with R15F01-del1 fragment, but lacZ CDS contains a frameshift mutation
Genetic reagent (Drosophila melanogaster)	R15F01-del2-lacZ(attP40)	this paper	N/A	lacZ reporter with R15F01-del2 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-del3-lacZ(attP40)	this paper	N/A	lacZ reporter with R15F01-del3 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-del4-lacZ(attP40)	this paper	N/A	lacZ reporter with R15F01-del4 fragment
Genetic reagent (Drosophila melanogaster)	trh47-GAL4(attP2)	this paper	N/A	GAL4 transgene with trh47 regulatory region
Genetic reagent (Drosophila melanogaster)	trh67-GAL4(attP2)	this paper	N/A	GAL4 transgene with trh67 regulatory region
Genetic reagent (Drosophila melanogaster)	R15F01-GAL4(attP40)	this paper	N/A	GAL4 transgene with R15F01 fragment
Genetic reagent (Drosophila melanogaster)	R14E10-GAL4(attP40)	this paper	N/A	GAL4 transgene with R14E10 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-D1-GAL4(attP2)	this paper	N/A	GAL4 transgene with R15F01-D1 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-D7-GAL4 (attP2)	this paper	N/A	GAL4 transgene with R15F01-D7 fragment
Genetic reagent (Drosophila melanogaster)	R15F01-del4-GAL4 (attP2)	this paper	N/A	GAL4 transgene with R15F01-del4 fragment
Genetic reagent (Drosophila melanogaster)	svbE6B-lacZ(attP40)	this paper	N/A	lacZ reporter with svbE6B regulatory region, integrated into attP40
Genetic reagent (Drosophila melanogaster)	svbE6B-D3-6-lacZ(attP40)	this paper	N/A	lacZ reporter with svbE6B-R15F01-D3-6 fusion fragment
Genetic reagent (Drosophila melanogaster)	svbE6B-lacZ(ZH-51C)	this paper	N/A	lacZ reporter with svbE6B regulatory region, integrated into ZH-51C
Genetic reagent (Drosophila melanogaster)	svbE6B-D1-2-lacZ(ZH-51C)	this paper	N/A	lacZ reporter with svbE6B-R15F01-D1-2 fusion fragment
Genetic reagent (Drosophila melanogaster)	svbE6B-del1-lacZ(ZH-51C)	this paper	N/A	lacZ reporter with svbE6B-R15F01-del1 fusion fragment
Genetic reagent (Drosophila melanogaster)	3×UAS-GAP43GFP (attP2)	this paper	N/A	GFP reporter with3 × UAS sequences
Genetic reagent (Drosophila melanogaster)	3×UAS-R15F01-GAP43GFP (attP2)	this paper	N/A	GFP reporter with 3 × UAS-R15F01 fusion
Antibody	anti-β-galactosidase	MP Biomedicals	55976, RRID:AB_2334934	rabbit polyclonal, 1:5000 for IHC
Antibody	anti-β-galactosidase	Abcam	ab9361, RRID:AB_307210	chick polyclonal, 1:1000 for IHC
Antibody	anti-DE-cad	Developmental Studies Hybridoma Bank	DSHB: DCAD2, RRID:AB_528120	rat monoclonal, 1:20 for IHC
Antibody	anti-RFP	MBL	M155-3, RRID:AB_1278880	mouse monoclonal, 1:300 for IHC
Antibody	anti-DsRed	BD Biosciences	632397	rabbit polyclonal, 1:5000 for IHC
Antibody	anti-GFP	Molecular Probes	A-11122, RRID:AB_221569	rabbit polyclonal, 1:300 for IHC
Antibody	anti-Trh	other		rabbit polyclonal, 1:100 for IHC, Dr Jordi Casanova (IRB Barcelona)
Antibody	Anti-MAP Kinase, Activated	Signa-Aldrich	M8159, RRID:AB_477245	mouse monoclonal, 1:1000 for IHC
Antibody	anti-rabbit IgG Alexa Fluor Plus 488	Molecular Probes	A-32731, RRID:AB_2633280	1:300 for IHC
Antibody	anti-rabbit IgG Alexa Fluor Plus 555	Molecular Probes	A-32732, RRID:AB_2633281	1:300 for IHC
Antibody	anti-mouse IgG Alexa Fluor Plus 488	Molecular Probes	A-32723, RRID:AB_2633275	1:300 for IHC
Antibody	anti-mouse IgG Alexa Fluor Plus 555	Molecular Probes	A-32727, RRID:AB_2633276	1:300 for IHC
Antibody	anti-rabbit IgG Alexa Fluor 488	Molecular Probes	A-11034, RRID:AB_2576217	1:300 for IHC
Antibody	anti-rabbit IgG Alexa Fluor 555	Molecular Probes	A-21429, RRID:AB_141761	1:300 for IHC
Antibody	anti-mouse IgG Alexa Fluor 488	Molecular Probes	A-11029, RRID:AB_138404	1:300 for IHC
Antibody	anti-mouse IgG Alexa Fluor 555	Molecular Probes	A-21424, RRID:AB_141780	1:300 for IHC
Antibody	anti-Rat IgG DyLignt 649	Jackson ImmunoResearch	112-495-167	1:300 for IHC
Antibody	anti-Rat IgG DyLignt 650	Abcam	ab102263, RRID:AB_10711247	1:50 for IHC
Antibody	anti-chick IgY Alexa Fluor 488	Abcam	ab150173	1:300 for IHC
Antibody	anti-chick IgY Alexa Fluor 555	Abcam	ab150174	1:300 for IHC
Antibody	anti-mouse IgG-biotin	Jackson ImmunoResearch	715-065-151, RRID:AB_2340785	1:500 for TSA amplification
Commercial assay or kit	VECTASTAIN Universal Elite ABC Kit	Vector Laboratories	PK-6100	Use Reagent A and Reagent B for TSA amplification
Commercial assay or kit	TSA Cyanine 3 System	PerkinElmer	NEL704A001KT	1:50 for TSA amplification