rabbit anti c fos antibody  (Cell Signaling Technology Inc)

Journal: Advanced Science

Article Title: Cannabinoid CB1 Receptor in Nociceptors Mediates Postoperative Analgesia via ASIC3 Inhibition

doi: 10.1002/advs.77925

Figure Lengend Snippet: ASIC3‐expressing nociceptors are activated in the DRG and contribute to incision‐induced postoperative pain. (A–C) Representative c‐Fos immunostaining images and quantification in WT DRG sections on postoperative day 1. Scale bar = 20 µm, 20 × objective, zoom = 1.97 ×. Unpaired t tests; n = 4 mice/group, 3–4 sections/mouse. (D–F) Western blot analysis of ASIC3 expression in WT mouse DRG and spinal dorsal horn (SDH) on postoperative day 1. Unpaired t tests; n = 5 mice/group. (G,H) ASIC3 protein levels in the DRG of littermate WT mice and Trpv1 ; Cnr1 fl/fl mice on day 1 post‐incision. Unpaired t tests; n = 5 mice/group. (I) i.pl. injection of the selective ASIC3 blocker APETx2 on postoperative day 1 robustly reverses mechanical allodynia in WT mice. (J) PWT following i.pl. injection of the ASIC3 blocker APETx2 in naïve Trpv1 ; Cnr1 fl/fl mice. Two‐way ANOVA with Sidak's test for multiple comparisons; * p < 0.05, ** p < 0.01. n = 6–11 mice/group; means ± SEM. M, male; F, female; BL, baseline; Veh, vehicle.

Article Snippet: The sections were blocked for 1 h in 5% donkey serum (SL050, Solarbio) with 0.1% Triton X‐100 and incubated overnight at 4°C with primary antibodies against ASIC3 (ASC‐018, Alomone), c‐Fos (226017, SYSY), TRPV1 (ab6166, Abcam), CGRP (ab47027, Abcam), IB4 (L2895, Sigma), NF200 (N0142, Sigma), and pERK 1/2 (9101S, Cell Signaling Technology) at 4°C overnight.

Techniques: Expressing, Immunostaining, Western Blot, Analysis, Injection

Journal: Science Advances

Article Title: Kappa opioids suppress histaminergic itch by inhibiting mediodorsal thalamus-projecting claustrum glutamatergic neurons

doi: 10.1126/sciadv.ady4282

Figure Lengend Snippet: ( A ) Schematic of histamine injection and experimental timeline. ( B ) Increased scratching bouts induced by intradermal injection of histamine in mice. **** P < 0.0001, Student’s t test, n = 5 to 6 mice. ( C and D ) Histamine-induced itch increased c-fos expression in multiple brain regions. (C) Representative images and (D) quantification of c-fos cells per mm 2 in saline and histamine-treated mice. Scale bar, 100 μm. * P < 0.05, ** P < 0.01, Student’s t test, n = 4 to 7 mice. All data are shown as mean ± SEM. PFC, prefrontal cortex; CLA, claustrum; PV, paraventricular nucleus; PBN, parabrachial nucleus.

Article Snippet: C-fos antibody (rabbit anti c-fos) (226003, RRID: AB_2231974) was purchased from Synaptic Systems Company.

Techniques: Injection, Expressing, Saline

Journal: Science Advances

Article Title: Kappa opioids suppress histaminergic itch by inhibiting mediodorsal thalamus-projecting claustrum glutamatergic neurons

doi: 10.1126/sciadv.ady4282

Figure Lengend Snippet: ( A and B ) Histamine-induced c-fos expression within CLA colocalized with vGlut1 mRNA, but not with GAD65 mRNA. (A) Left: Representative images, including c-fos (white), vGlut1 (green) and DAPI (blue); Right: Quantitative analysis (per mm 2 ). Scale bar, 50 μm. n = 3 mice. (B) Left: Representative images of the CLA, including c-fos (white), GAD65 (red) and DAPI (blue); Right: Quantitative analysis (per mm 2 ). Scale bar, 50 μm. n = 3 mice. ( C ) Schematic diagram and experimental procedure. ( D ) Representative image. Scale bar, 200 μm. ( E to H ) Representative traces of Ca 2+ signals of CLA glutamatergic neurons. (E) Synchronization of Ca 2+ signals with markers of scratching behavior. (F) Representative traces of Ca 2+ signals (red line) during scratching occur (−10 s − +10 s). n = 4 mice. (G) Heatmaps across animals aligned to the beginning of scratching. (H) Quantification of the area under the curve (AUC, −10–0 s, 0 s–10 s) of average Δ F/F . ** P < 0.01. Student’s t test. n = 4 mice. ( I ) Schematic diagram and experimental procedure. ( J and M ) Representative images of c-fos cells among CLA glutamatergic neurons. Scale bar, 50 μm, 20 μm. (J) The inhibitory effects of the hM4Di. (M) The excitatory effects of the hM3Dq. ( K and N ) Quantification of c-fos and mCherry co-labeled cells. (K) ** P < 0.01. Student’s t test, n = 3 mice. (N) *** P < 0.001. Student’s t test, n = 3 mice. ( L ) Chemogenetic inhibition of CLA glutamatergic neurons decreased histamine-induced scratching behavior. * P < 0.05. Student’s t test, n = 15 to 16 mice. ( O ) Chemogenetic activation of CLA glutamatergic neurons increased low-dose histamine-induced scratching behavior. * P < 0.05. Student’s t test, n = 8 to 9 mice. All data are shown as mean ± SEM.

Article Snippet: C-fos antibody (rabbit anti c-fos) (226003, RRID: AB_2231974) was purchased from Synaptic Systems Company.

Techniques: Expressing, Labeling, Inhibition, Activation Assay

Journal: Science Advances

Article Title: Kappa opioids suppress histaminergic itch by inhibiting mediodorsal thalamus-projecting claustrum glutamatergic neurons

doi: 10.1126/sciadv.ady4282

Figure Lengend Snippet: ( A and B ) Schematic diagram of experimental procedure. ( C ) Systemic injection of Nalfurafine and U50488H decreased histamine-induced scratching behavior. ** P < 0.01, **** P < 0.0001. Student’s t test, n = 5 to 6 mice. ( D and E ) Nalfurafine and U50488H decreased histamine-induced c-fos expression in CLA. (D) Representative images and (E) quantification of c-fos cells in CLA. Scale bar, 50 μm. * P < 0.05, ** P < 0.01, One-way ANOVA, n = 5 mice. ( F and I ) Schematic diagram of experimental procedure. ( G ) Microinjection of Nalfurafine and U50488H in the CLA decreased histamine-induced scratching behavior. ** P < 0.01, **** P < 0.0001. Student’s t test, n = 6 to 10 mice. ( H ) Schematic representation of Nalfurafine and U50488H microinjection sites in the CLA. ( J ) Microinjection of nor-BNI into the CLA blocked the antipruritic effects of systemically administered Nalfurafine and U50488H. *** P < 0.001, **** P < 0.0001, One-way ANOVA, n = 8 to 12 mice. ( K ) Schematic representation of nor-BNI microinjection sites in the CLA. All data are shown as mean ± SEM.

Article Snippet: C-fos antibody (rabbit anti c-fos) (226003, RRID: AB_2231974) was purchased from Synaptic Systems Company.

Techniques: Injection, Expressing, Microinjection

Journal: Science Advances

Article Title: Kappa opioids suppress histaminergic itch by inhibiting mediodorsal thalamus-projecting claustrum glutamatergic neurons

doi: 10.1126/sciadv.ady4282

Figure Lengend Snippet: ( A ) Anterograde viral tracing from CLA to MD: schematic and representative fluorescence images. ( B ) Schematic and representative fluorescence images of retrograde viral tracing from CLA to MD. Scale bar, 100 μm. ( C to E ) Histamine-induced itch increased c-fos expression in the MD. (C) Schematic diagram of experimental procedure. (D) Representative images and (E) quantification of c-fos cells. Scale bar, 200 μm. * P < 0.05. Student’s t test, n = 8 mice. ( F to I ) MD glutamatergic neurons exhibit significant activation. (F) Schematic diagram and representative image and experimental procedure. Scale bar, 100 μm. (G to I) Representative traces of Ca 2+ signals of MD glutamatergic neurons. (G) Representative traces of Ca 2+ signals (blue line) during scratching occur (−10 s − +10 s). n = 3 mice. (H) Heatmaps across animals aligned to the beginning of scratching. (I) Quantification of the AUC (−10–0 s, 0 s–10 s) of average Δ F/F . *** P < 0.001. Student’s t test, n = 3 mice. ( J to M ) Axon terminals of CLA Glu neurons projecting to the MD exhibit significant activation during histamine-induced scratching behavior. (J) Schematic diagram and representative image and experimental procedure. Scale bar, 100 μm. (K) Representative traces of Ca 2+ signals (brown line) during scratching occur (−10 s − +10 s). n = 3 mice. (L) Heatmaps across animals aligned to the beginning of scratching. (M) Quantification of the AUC (−10–0 s, 0 s–10 s) of average Δ F/F . *** P < 0.001. Student’s t test, n = 3 mice. All data are shown as mean ± SEM.

Article Snippet: C-fos antibody (rabbit anti c-fos) (226003, RRID: AB_2231974) was purchased from Synaptic Systems Company.

Techniques: Fluorescence, Expressing, Activation Assay