RNAscope Multiplex Fluorescent V2 assay (Bio-techne, Cat#:323110) was
performed according to manufacturer’s instruction on 4 μm paraffin
sections, hybridized with the probes Mm-Gpbar1-C1(Bio-techne, Cat#:318451),
Mm-Agrp-C2 (Bio-techne, Cat#:400711-C2), Mm-Ppib-C1 (Bio-techne, Cat#:313911) as
positive control and DapB-C1 (Bio-techne, Cat#:310043) as negative control at
40°C for 2 hours and revealed with TSA Plus Cy3 (Perkin Elmer,
Cat#:NEL744E001KT). Tissues were counterstained with DAPI and mounted with
FluoromountG (Bioconcept, Cat#:100.01). When indicated, sections were incubated
overnight at 4°C with a goat anti-GFP primary antibody (Abcam,
Cat#:ab6673 – 1:200 dilution). After incubation with a donkey anti-goat
Alexa488 secondary antibody (Thermo Fisher, Cat#:A11055, 1:1000 dilution),
tissues were counterstained with DAPI and mounted with FluoromountG (Bioconcept,
Cat#:100.01). The imaging was performed with Zeiss LSM 700 confocal microscope
(Carl Zeiss Microscopy, Germany).
performed according to manufacturer’s instruction on 4 μm paraffin
sections, hybridized with the probes Mm-Gpbar1-C1(Bio-techne, Cat#:318451),
Mm-Agrp-C2 (Bio-techne, Cat#:400711-C2), Mm-Ppib-C1 (Bio-techne, Cat#:313911) as
positive control and DapB-C1 (Bio-techne, Cat#:310043) as negative control at
40°C for 2 hours and revealed with TSA Plus Cy3 (Perkin Elmer,
Cat#:NEL744E001KT). Tissues were counterstained with DAPI and mounted with
FluoromountG (Bioconcept, Cat#:100.01). When indicated, sections were incubated
overnight at 4°C with a goat anti-GFP primary antibody (Abcam,
Cat#:ab6673 – 1:200 dilution). After incubation with a donkey anti-goat
Alexa488 secondary antibody (Thermo Fisher, Cat#:A11055, 1:1000 dilution),
tissues were counterstained with DAPI and mounted with FluoromountG (Bioconcept,
Cat#:100.01). The imaging was performed with Zeiss LSM 700 confocal microscope
(Carl Zeiss Microscopy, Germany).