3. Periodically capture z-stacks of the prepupa on an inverted
confocal in a room with temperature kept at 25
C, or with a
stage heater. For us, a typical imaging setup is a Nikon A1R
inverted confocal, 20Â PL APO Objective, 512 Â 512 pixels,
5–10 slices of 3 μm, taken every 60–90 s (see Note 12).
3.5 Immunohistochemistry of the
Peripodial pEMT
In Vivo
1. Remove prepupae from the vial by dislodging them gently with
a paintbrush or using forceps to take hold of the anterior
spiracles and place them in the fixative solution of 4% formaldehyde in PBST. Take care not to puncture or deform them.
Dislodge any food by holding them with forceps and using a
paintbrush. Remove floating debris with a P1000 tip and
replenish the fixative solution.
2. Using scissors, cut the tips off the posterior spiracles to allow
the fixative to enter and stabilize the internal tissues. Then use
two sharp forceps to pull apart the pupal case, anterior to the
midpoint (0.3–0.4 of the pupal length, from the anterior),
separating it into anterior and posterior halves, taking care
not to deform the pupal case. Fix for 20 min (see Note 13).
3. While fixation is proceeding, using forceps, remove as much of
the internal tissue (i.e., fat body, gut, salivary glands) as possible, without disturbing the dorso-lateral wing disc region (see
Note 14). One can further increase the accessibility of the wing
disc area by cutting the anterior half with micro-scissors, into
dorsal (containing the wing discs) and ventral parts. This will
also further facilitate removal of unwanted tissue.
4. Wash off the fixative 3–4 times with PBST3.
5. Immunostaining of the pupal tissue is then carried out using
standard methods—but with base solution of PBST3, unless
using the DCAD2 antibody in which case PBS is replaced
with PB.
3.6 Ex Vivo Eversion
The protocol for ex vivo eversion is as described in Aldaz et al.
[14]. We use ex vivo eversion in two assays (Fig. 1). The first is a
simple overnight assay, whereby discs are given enough time to fully
evert if they are able, and then scored as having everted successfully,
partially or not at all (Fig. 3). The other assay is to culture the discs
up to the time of the onset of the pEMT (~7–8 h) and then fix and
stain them (Fig. 4).
1. Select wandering third-instar larvae and transfer them to a
cavity block with PBS. Wash off food and excess debris and
then transfer larvae to 70% ethanol for 5 min to disinfect them.
Wash again in PBS, and transfer to a cavity block containing
culture medium.
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Sofia Golenkina et al.
confocal in a room with temperature kept at 25
C, or with a
stage heater. For us, a typical imaging setup is a Nikon A1R
inverted confocal, 20Â PL APO Objective, 512 Â 512 pixels,
5–10 slices of 3 μm, taken every 60–90 s (see Note 12).
3.5 Immunohistochemistry of the
Peripodial pEMT
In Vivo
1. Remove prepupae from the vial by dislodging them gently with
a paintbrush or using forceps to take hold of the anterior
spiracles and place them in the fixative solution of 4% formaldehyde in PBST. Take care not to puncture or deform them.
Dislodge any food by holding them with forceps and using a
paintbrush. Remove floating debris with a P1000 tip and
replenish the fixative solution.
2. Using scissors, cut the tips off the posterior spiracles to allow
the fixative to enter and stabilize the internal tissues. Then use
two sharp forceps to pull apart the pupal case, anterior to the
midpoint (0.3–0.4 of the pupal length, from the anterior),
separating it into anterior and posterior halves, taking care
not to deform the pupal case. Fix for 20 min (see Note 13).
3. While fixation is proceeding, using forceps, remove as much of
the internal tissue (i.e., fat body, gut, salivary glands) as possible, without disturbing the dorso-lateral wing disc region (see
Note 14). One can further increase the accessibility of the wing
disc area by cutting the anterior half with micro-scissors, into
dorsal (containing the wing discs) and ventral parts. This will
also further facilitate removal of unwanted tissue.
4. Wash off the fixative 3–4 times with PBST3.
5. Immunostaining of the pupal tissue is then carried out using
standard methods—but with base solution of PBST3, unless
using the DCAD2 antibody in which case PBS is replaced
with PB.
3.6 Ex Vivo Eversion
The protocol for ex vivo eversion is as described in Aldaz et al.
[14]. We use ex vivo eversion in two assays (Fig. 1). The first is a
simple overnight assay, whereby discs are given enough time to fully
evert if they are able, and then scored as having everted successfully,
partially or not at all (Fig. 3). The other assay is to culture the discs
up to the time of the onset of the pEMT (~7–8 h) and then fix and
stain them (Fig. 4).
1. Select wandering third-instar larvae and transfer them to a
cavity block with PBS. Wash off food and excess debris and
then transfer larvae to 70% ethanol for 5 min to disinfect them.
Wash again in PBS, and transfer to a cavity block containing
culture medium.
126
Sofia Golenkina et al.