rnascope probe-mm-adcyap1  (Advanced Cell Diagnostics Inc)

Journal: Research Square

Article Title: Prolonged activation of EP3 receptor-expressing preoptic neurons underlies torpor responses

doi: 10.21203/rs.3.rs-2861253/v1

Figure Lengend Snippet: RNA-scope in situ hybridization (ISH) showed the overlap of EP3R (red, Ptger3 gene) and PACAP (blue, Adcyap1 gene) in the MnPO of a Vglut2-IRES-Cre-L10 mouse (green, Slc17a6 ) (a). The colocalization of Ptger3, Adcyap1 and Slc17a6 is clear at a higher magnification (b-e). We found about 45% of the MnPO PACAP neurons co-expressing EP3R and 61% of EP3R neurons expressing PACAP. We crossed Ptger3 flox/flox mice with Adcyap1 Cre mice to cause deletion of EP3R from PACAP+ neurons (PACAP+EP3R-null mice) (f). ISH showed successful deletion of EP3R (green) from MnPO PACAP (red) neurons, and a remaining subset of MnPO EP3R+/PACAP- neurons (white arrows) (g-i). In WT mice, after the first peak of Tb elevation caused by the stress, mice showed a typical fever response 1–3h after LPS injection. However, in PACAP+EP3R-null mice LPS caused a small hypothermic response 1–3 hr after injection (0.4 °C ± 0.03 WT SAL vs. 1.1 °C ± 0.04 WT LPS vs. 0.03 °C ± 0.04 PACAP+EP3R-null mice SAL vs. −0.4 °C ± 0.04 PACAP+EP3R-null mice LPS, F(3,92)=195.1 One-way ANOVA, followed by Bonferroni’s post hoc test, p<0.0001) (j-k). A schematic figure of the protocol for calcium imaging (left panel) and a representative image of GCAMP6s native fluorescence expression and fiber placement in a Adcyap1 Cre male mouse is shown (right panel) (l). A recording of calcium signaling (green) and Tb (black) of a Adcyap1 Cre mouse illustrates a reduction in fluorescence during fever relative to the baseline (DF/F) (m). Data analyses of 1-h bins of the DF/F before and after LPS injection normalized by the baseline condition show a robust reduction in the DF/F of calcium fluorescence in MnPO PACAP neurons during fever (n=3), and standard variance (SD, i.e., spikiness) of the signal peaks before fever is reduced after the LPS injection (n-o). These results suggest that fever may be caused by the inhibition of hypothermic MnPO PACAP+ neurons. To test this, we photoinhibited MnPO PACAP neurons (p). Photoinhibiting MnPO PACAP cell bodies for 15 min (10 stimuli of 60 s on followed by 30 s off) caused a hyperthermic effect that lasted for ~ 1 h (n=4) compared to sham stimulation (0.6 ± 0.09°C SEM MnPO STIM vs 0.06 ± 0.04 °C SEM SHAM STIM, paired t-test, p<0.0001) (q). ArchT-GFP+ expression and optical fiber placement in MnPO neurons of a Adcyap1 Cre mouse (r).

Article Snippet: POA sections were used for Ptger3 (EP3R) mRNA labelling (RNAscope Probe- Mm- Ptger3 ; catalog #504481, Advanced Cell Diagnostics) or Adcyap1 (PACAP) mRNA labelling (RNAscope Probe-Mm-Adcyap1; catalog #405911-C2, Advanced Cell Diagnostics, CA).

Techniques: RNAscope, In Situ Hybridization, Expressing, Injection, Imaging, Fluorescence, Inhibition

Journal: eLife

Article Title: Neuroendocrinology of the lung revealed by single-cell RNA sequencing

doi: 10.7554/eLife.78216

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , RNAscope Hiplex assay probe Mm-Adcyap1-T3 , Advanced Cell Diagnostics (ACD) , Cat#405911-T3 , NA.

Techniques: RNAscope, In Situ Hybridization, Software