polyclonal goat anti paired box 2  (R&D Systems)

Journal: bioRxiv

Article Title: Descending locus coeruleus noradrenergic signaling to spinal astrocyte subset is required for stress-induced pain facilitation

doi: 10.1101/2024.11.14.623627

Figure Lengend Snippet: (A and B) Intrathecal NA (A, 0.1 nmol) or Phe (B, 0.05 nmol)-induced mechanical hypersensitivity in control ( Adra1a flox/flox ) and Vgat + IN-selective α 1A R conditional knockout mice [ Vgat-Cre ; Adra1a flox/flox mice (Vgat + INs–α 1A R cKO mice)] (n = 6 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; n.s., not significant). (C) Changes in PWT after acute exposure to restraint stress in control ( Adra1a flox/flox ; n = 6 mice) and Vgat + INs–α 1A R cKO mice ( n = 9 mice) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test). (D) Representative trace of membrane potentials in tdTomato + (Vgat + ) SDH neurons after application of NA (20 μM) to spinal cord slices from Vgat-Cre ; Rosa-tdTomato mice. A 1 R agonist CPA (1 μM) was co-applied with NA. (E) Percentage of Vgat + SDH neurons whose NA-evoked response was inhibited by CPA ( n = 10 cells from 7 mice). (F) Representative images of Adora1 (A 1 R) mRNA expression (green) in Slc32a1 (Vgat) + INs (magenta). DAPI staining is shown in gray. Arrowheads indicate A 1 R-expressing Vgat + cells. Scale bar, 25 μm. (G) SaCas9 (yellow, detected by HA-tag) and mCherry (magenta) expression in the PAX2 + INs (cyan) at 3 weeks after intra-SDH injection of AAV-flex[SaCas9] and AAV-flex[mCherry]-U6-sgAdora1 in Vgat-Cre mice. Arrowheads indicate genome-editing Vgat + cells. Scale bar, 25 μm. (H) Representative traces of membrane potentials in Vgat + INs after application of NA and CPA to spinal cord slices from Vgat-Cre mice with conditional knockdown of A 1 Rs in Vgat + INs (SDH-Vgat + IN–A 1 R cKD) and their controls (Control; Vgat-Cre mice with intra-SDH injection of AAV-flex[mCherry]). (I) Percentage of mCherry + (Vgat + ) SDH neurons whose NA-evoked response was inhibited by CPA (Control, n = 10 cells from 8 mice; SDH-Vgat + IN–A 1 R cKD, n = 8 cells from 5 mice). (J) Expression of hM3Dq (green, detected by HA-tag) in the SDH at 3 weeks after intra-SDH injection of AAV-flex[hM3Dq] in Hes5-CreERT2 mice treated with TAM. Dashed line indicates an outline of the gray matter of SDH. GFAP (magenta, bottom left), SOX9 (magenta, bottom right). Arrowheads indicate HA-tag + astrocytes. Scale bars, 200 μm (top) and 25 μm (bottom). (K and L) Representative traces of sIPSCs (K) and quantitative analysis of their frequency (L) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-hM3Dq mice treated with TAM [Pre and CNO: before and after bath application of CNO (100 μM), respectively] ( n = 7 cells from 7 mice; Wilcoxon signed-rank test; * P < 0.05). (M and N) Representative traces of sIPSCs (M) and quantitative analysis of their frequency (N) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-flex[hM3Dq] mice treated with TAM [Pre and CNO: before and after bath application of CNO with CPT (1 μM), respectively] ( n = 13 cells from 13 mice; Wilcoxon signed-rank test; n.s., not significant). Data represent mean ± SEM.

Article Snippet: Transverse spinal cord and brain sections (30 μm) were incubated in blocking solution (3% normal goat serum [#S-1000; Vector Laboratories] or normal donkey serum [#017-000-121; Jackson ImmunoResearch]) for 2 hours at room temperature and then incubated for 48 hours at 4°C with primary antibodies: polyclonal rabbit anti-tyrosine hydroxylase (TH; 1:1000; #AB152; Millipore); polyclonal sheep anti-TH (1:1000; #AB1542; Millipore); monoclonal mouse anti-noradrenaline transporter (NET; 1:2000; #NET05-2; Mab Technologies); polyclonal rabbit anti-green fluorescent protein (GFP; 1:1000; #598; MBL International); monoclonal rabbit anti-hemagglutinin (HA)-tag (1:1000; #3724; Cell Signaling); monoclonal rat anti-glial fibrillary acidic protein (GFAP; 1:2000; #13-0300; Invitrogen); polyclonal goat anti-SRY-related high-mobility group box 9 (SOX9; 1:1000; # AF3075; R&D Systems); polyclonal goat anti-paired box 2 (PAX2; 1:500; # AF3364; R&D Systems); monoclonal rat anti-mCherry (1:2000; #M11217; Thermo Fisher Scientific); polyclonal rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (pERK; 1:500; #9101; Cell Signaling); anti-isolectin B4 (IB4)-biotin conjugate (1:1000; # I21414; Thermo Fisher Scientific).

Techniques: Control, Knock-Out, Membrane, Expressing, Staining, Injection, Knockdown