2.3. FastBlue tracer injections
Mice were briefly anesthetized during the procedure, induced with
3% to 5% isoflurane, and then maintained at 1.5% to 2% as required.
Hindlimbs were taped with the plantar surface of the paw facing up,
and a custom, 26G removable needle with a 30˚ bevel, attached to
a2 5 -mL Hamilton syringe, was inserted between the 2 distal-most
footpads, towards the medial aspect of the hindpaw. The needle
was then rotated 90˚, so the bevel faced medially. Furthermore, 4-mL
FastBlue (FB; 2% in sterile phosphate-buffered saline (PBS); CAS#
73819-41-7; Polysciences, Inc, Warrington, PA) per paw was then
slowly injected, and the needle was left in place for 10 seconds,
before rotating and carefully retracting to avoid backflow of FB along
the needle track. This prevented the FB bolus from contacting the
sural innervation territory of the lateral hindpaw, restricting it largely to
the tibial innervation territory of the glabrous hindpaw skin.
2.4. Immunohistochemistry and image acquisition
Mice were anesthetized with an overdose of pentobarbital (20
mg) and transcardially perfused with a fixative containing 4%
formaldehyde. L3 to L5 DRGs were removed and postfixed for
another 2 hours, cryoprotected in 30% sucrose overnight, and
then embedded in optimal cutting temperature media (OCT;
Tissue Tek, Alphen aan den Rijn, the Netherlands). Dorsal root
ganglia were sectioned on a Leica CM1950 cryostat at 30mm,
with every section collected serially on 5 Superfrost Plus slides
(VWR, Lutterworth, United Kingdom) and each slide containing 1
in every 5 sections (4-7 sections per slide). One slide per DRG was
selected at random and was washed with PBS, before being
incubated with appropriate primary antibodies (Table 2) diluted in
5% normal donkey serum and 0.3% Triton X-100 in PBS for
3 days at 4˚C. After PBS washes, slides were incubated with
appropriate secondary antibodies ( Table 2) in the same PBS/
(normal donkey serum) NDS/Triton-X100 solution as for prima-
ries, overnight at room temperature. Slides were washed and
coverslipped with VectaShield Vibrance Hardset mounting media
(Vector Labs, Newark, CA), with 4’,6-diamidino-2-phenylindole
included in mounting media where FB-labelled cells were not
being examined. Sections were imaged using a Zeiss LSM900
Airyscan confocal microscope equipped with 405-, 488-, 561-,
Table 2
Primary and secondary antibodies used in the study.
Antibody Source Identifiers Working dilution
Anti-GFP (Chicken polyclonal) Abcam, plc, Cambridge, United Kingdom Cat#: ab13970
RRID: AB_300798
1:1000
Anti-NeuN (Guinea pig polyclonal) Synaptic Systems, G ¨ ottingen, Germany Cat#: 266004
RRID: AB_2619988
1:500
Anti-mCherry (Rat monoclonal) Invitrogen, Waltham, MA; Thermo Fisher Scientific,
United Kingdom
Cat#: M11217
RRID: AB_2536611
1:500
Anti-Atf3 (Rabbit polyclonal) Novus Biologicals, Minneapolis, MN Cat#: NBP1-85816
RRID: AB_11014863
1:500
Anti-NF200 (Rabbit polyclonal) Sigma-Aldrich, Saint Louis, MO Cat#: N4142
RRID: AB_477272
1:1000
Anti-TrkA (Goat polyclonal) R&D Systems, Minneapolis, MN Cat#: AF1056
RRID: AB_2283049
1:500
Anti-TDP43 (Rabbit polyclonal) Abcam, plc, Cambridge, United Kingdom Cat#: ab133547
RRID: AB_2920621
1:100
Anti-RFP (Mouse monoclonal) Thermo Fisher Scientific, United Kingdom Cat#: MA5-15257
RRID: AB_10999796
1:200
Anti-RFP (Chicken polyclonal) Sigma-Aldrich, United Kingdom Cat#: AB3528
RRID: AB_11212735
1:200
Alexa Fluor 488 Donkey Anti-Chicken IgY
(Donkey polyclonal)
Jackson ImmunoResearch, Ely, United Kingdom Cat#: 703-545-155
RRID: AB_2340375
1:500
Alexa Fluor 647 Donkey Anti-Guinea pig IgG
(Donkey polyclonal)
Jackson ImmunoResearch, Ely, United Kingdom Cat#: 706-605-148
RRID: AB_2340476
1:250
Rhodamine Red-X Donkey Anti-Rat IgG (Donkey
polyclonal)
Jackson ImmunoResearch, Ely, United Kingdom Cat#: 712-295-153
RRID: AB_2340676
1:100
Alexa Fluor 647 Donkey Anti-Rabbit IgG (Donkey
polyclonal)
Jackson ImmunoResearch, Ely, United Kingdom Cat#: 711-605-152
RRID: AB_2492288