10. PBST: 1Â PBS, 0.1% Triton X.
11. PBST3: 1Â PBS + 0.3% Triton X.
12. 10Â PB ¼ 2 M Na 2 HPO 4 , 2 M NaH 2 PO 4 , pH 7.4.
2.3 Antibodies
and Stains for EMT
Factors
2.3.1 Adherens
Junctions
l Rat-anti-DECadherin
DCAD2, Developmental Studies Hybridoma Bank
(DSHB)
l Rat-anti-alphaCatenin
DCAT-1, DSHB
l Mouse-antiarmadillo
N27A1, DSHB
2.3.2 F-Actin
l Rhodamine-Phalloidin
PHDR1, Cytoskeleton, Inc.
l Phalloidin-iFluor 488 (and spectral variants) ab176753, Abcam
2.3.3 JNK-Pathway
Activation
l Phospho-JNK
9251, Cell Signaling Technology
l Mouse-anti-MMP1
3A6B4, DSHB
2.3.4 Frazzled
Localization
l Rabbit-anti-Frazzled
Antibody to the C-terminal domain [41]
(Available from M.J.M. and others)
2.3.5 Basement
Membrane
Although we and others have used previously a commercial antibody that recognized Drosophila LanB1 (Abcam Rabbit-anti-laminin β1 ab47650), it is no longer available, and we are unaware of
other commercial antibodies that recognize Drosophila ECM components (i.e., Collagen IV, Laminin, Nidogen, Perlecan). Antibodies for Perlecan [43], Nidogen [44, 45], and laminins, e.g., [46]
should be requested from the appropriate researchers. Our group
has confirmed that, like Coll IV and Lan B1, Nidogen is present in
the BM of the wing discs.
2.4 Equipment
for Pupal Dissections
and Tissue Culture
1. Molded glass cavity/staining blocks with hemispherical cavity.
2. Two pairs of sharp forceps.
3. Vannas spring micro-scissors.
4. Paintbrush.
5. 90 mm petri dishes.
6. iBiDi culture dishes.
7. A medium sized (e.g., ~15 cm  15 cm  10 cm) plastic box to
act as a humidified chamber.
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11. PBST3: 1Â PBS + 0.3% Triton X.
12. 10Â PB ¼ 2 M Na 2 HPO 4 , 2 M NaH 2 PO 4 , pH 7.4.
2.3 Antibodies
and Stains for EMT
Factors
2.3.1 Adherens
Junctions
l Rat-anti-DECadherin
DCAD2, Developmental Studies Hybridoma Bank
(DSHB)
l Rat-anti-alphaCatenin
DCAT-1, DSHB
l Mouse-antiarmadillo
N27A1, DSHB
2.3.2 F-Actin
l Rhodamine-Phalloidin
PHDR1, Cytoskeleton, Inc.
l Phalloidin-iFluor 488 (and spectral variants) ab176753, Abcam
2.3.3 JNK-Pathway
Activation
l Phospho-JNK
9251, Cell Signaling Technology
l Mouse-anti-MMP1
3A6B4, DSHB
2.3.4 Frazzled
Localization
l Rabbit-anti-Frazzled
Antibody to the C-terminal domain [41]
(Available from M.J.M. and others)
2.3.5 Basement
Membrane
Although we and others have used previously a commercial antibody that recognized Drosophila LanB1 (Abcam Rabbit-anti-laminin β1 ab47650), it is no longer available, and we are unaware of
other commercial antibodies that recognize Drosophila ECM components (i.e., Collagen IV, Laminin, Nidogen, Perlecan). Antibodies for Perlecan [43], Nidogen [44, 45], and laminins, e.g., [46]
should be requested from the appropriate researchers. Our group
has confirmed that, like Coll IV and Lan B1, Nidogen is present in
the BM of the wing discs.
2.4 Equipment
for Pupal Dissections
and Tissue Culture
1. Molded glass cavity/staining blocks with hemispherical cavity.
2. Two pairs of sharp forceps.
3. Vannas spring micro-scissors.
4. Paintbrush.
5. 90 mm petri dishes.
6. iBiDi culture dishes.
7. A medium sized (e.g., ~15 cm  15 cm  10 cm) plastic box to
act as a humidified chamber.
122
Sofia Golenkina et al.