Immunohistology was performed as described previously (Weng et al., 2017 (link)). Briefly, samples
were collected from perfused animals, post-fixed overnight in 4% PFA
in PBS, and cryoprotected in 30% sucrose (wt/vol) for 24 hr at
4°C. Samples were sec tioned to 20 m and mounted onto slides.
Primary antibody was applied at 4°C overnight. Seco ndary antibody
was applied for 2 hr at room temperature. The following primary antibodies
were used in this study: rabbit anti-m6A (Synaptic Systems; 212B11; 1:2000),
rabbit anti-ATF3 (Santa Cruz; sc-188; 1:500), rabbit anti-PGP9.5 (AbD
Serotec; 7863-0504; 1:800), rabbit anti-SCG10 (Novus Biologicals;
NBP1-49461, 1: 2000), and anti-cleaved (active) form of caspase 3
(Invitrogen; 9H19L2; 1:500). Secondary antibodies corresponding to the
primary antibody species were Cy2–, Cy3– or Cy5 conjugated
(Jackson ImmunoResearch; 1:500). The images were acquired by confocal
microscopy (Zeiss 710) and analyzed with ImageJ software (National
Institutes of Health).
Quantification of the proportion of ATF3+ neurons
was determined by counting and scoring at least 200 neurons/mouse as
ATF3+ or ATF3−(Weng et al., 2017 (link)). A cell was
scored as ATF3+ if there was any fluorescence above the
threshold set in ATF3− cells under the
naïve conditions. Sections were randomly chosen from cross-sectioned
L4/L5 DRGs.
were collected from perfused animals, post-fixed overnight in 4% PFA
in PBS, and cryoprotected in 30% sucrose (wt/vol) for 24 hr at
4°C. Samples were sec tioned to 20 m and mounted onto slides.
Primary antibody was applied at 4°C overnight. Seco ndary antibody
was applied for 2 hr at room temperature. The following primary antibodies
were used in this study: rabbit anti-m6A (Synaptic Systems; 212B11; 1:2000),
rabbit anti-ATF3 (Santa Cruz; sc-188; 1:500), rabbit anti-PGP9.5 (AbD
Serotec; 7863-0504; 1:800), rabbit anti-SCG10 (Novus Biologicals;
NBP1-49461, 1: 2000), and anti-cleaved (active) form of caspase 3
(Invitrogen; 9H19L2; 1:500). Secondary antibodies corresponding to the
primary antibody species were Cy2–, Cy3– or Cy5 conjugated
(Jackson ImmunoResearch; 1:500). The images were acquired by confocal
microscopy (Zeiss 710) and analyzed with ImageJ software (National
Institutes of Health).
Quantification of the proportion of ATF3+ neurons
was determined by counting and scoring at least 200 neurons/mouse as
ATF3+ or ATF3−(Weng et al., 2017 (link)). A cell was
scored as ATF3+ if there was any fluorescence above the
threshold set in ATF3− cells under the
naïve conditions. Sections were randomly chosen from cross-sectioned
L4/L5 DRGs.