Immunohistochemistry was conducted on the tissue to enhance the fluorescence signal of mCherry (DREADD groups) or EGFP (control groups). The first series of sections was transferred from PBS into a blocking solution of 5% NGS in PBS with Triton X-1000 (PBST) and incubated for 1 h. The sections were then transferred into the primary antibody solution of rabbit-anti-mCherry or chicken polyclonal anti-GFP (Abcam) at a dilution of 1:1000 in PBST with 1% NGS and incubated for 24 h. Sections were washed 4 times in PBST and transferred to a secondary antibody solution of goat-anti-rabbit (Dylight AlexaFlour-594, Vector Laboratories) or goat-anti-chicken (Invitrogen) at a dilution of 1:200 at PBST. From this point onward, the sections were kept in the dark. Sections were incubated for 1 h before being washed 3 times in PBS. Sections were mounted onto gelatin-subbed glass slides and dried overnight before being immersed in xylene and coverslipped using DPX (Thermo Fisher Scientific). All incubations were on a stirring plate at room temperature, and all washes were for 10 min.
Dylight alexa flour 594
Manufactured by Vector Laboratories
Sourced in United Kingdom
DyLight Alexa Fluor 594 is a fluorescent dye used in various applications such as immunofluorescence, flow cytometry, and microscopy. It has an excitation maximum of 590 nm and an emission maximum of 617 nm, making it suitable for detection in the red/orange region of the visible spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Dibutylphthalate polystyrene xylene (dpx), by Thermo Fisher Scientific (4 mentions)
Dfc310 fx camera, by Leica camera (2 mentions)
Goat anti chicken, by Thermo Fisher Scientific (1 mentions)
Dm5000b, by Leica camera (1 mentions)
Rabbit anti ctb, by Merck Group (1 mentions)
Fluromount, by Merck Group (1 mentions)
Rabbit anti mcherry, by Abcam (1 mentions)
4 protocols using dylight alexa flour 594
1
Immunohistochemistry for mCherry and EGFP
2
Immunohistochemistry for mCherry Signal
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Immunohistochemistry was carried out on the tissue to enhance the fluorescence signal of mCherry (DREADD groups). The first series of sections was transferred from PBS into a blocking solution of 5% normal goat serum (NGS) in PBS with Tritonx-1000 (PBST) and incubated for 1 h. The sections were then transferred into the primary antibody solution of rabbit-anti-mCherry (Abcam) at a dilution of 1:1000 in PBST with 1% NGS and incubated for 24 h. Sections were washed 4 times in PBST and transferred to a secondary antibody solution of goat-anti-rabbit (Dylight Alexa flour 594, Vector Laboratories) or at a dilution of 1:200 at PBST. From this point onwards the sections were kept in the dark. Sections were incubated for 1 h before being washed 3 times in PBS. Sections were mounted onto gelatine-subbed glass slides and allowed to dry overnight before being immersed in xylene and coverslipped using DPX (Thermo Fisher Scientific). All incubations were on a stirring plate at room temperature and all washes were for 10 min. Virus expression was analyzed using a fluorescent Leica DM5000B microscope with a Leica DFC310 FX camera. Images were collected from the ACC, selected efferent targets, and a comparison cortical area, secondary somatosensory cortex.
3
Immunohistochemistry Staining Protocol for CTB
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For CTB staining, the sections were washed for 3 × 10 min in a .1‐M PBS, followed by 3 × 10 min washes in PBST (.2% Triton X‐100 in .1‐M PBS). Sections were then incubated with the primary antibody rabbit‐anti‐CTB overnight (1:3000) (Sigma‐Aldrich, UK) for 16–24 h at room temperature. The sections were then washed three times for 10 min with PBST and transferred to a secondary antibody of goat‐anti‐rabbit (Dylight Alexa flour 594, Vector Laboratories, Peterborough, UK) for 2 h on a stirrer. Finally, the sections were washed with PBS and mounted onto gelatin‐coated slides and cover‐slipped using Fluromount (Sigma‐Aldrich, Germany) or DPX (Thermo Fisher, Waltham, MA) mounting medium.
Where necessary to confirm the boundaries of the regions of interest, an additional series was mounted onto gelatin‐coated slides and Nissl‐stained using cresyl violet. The sections were then dehydrated through increasing concentrations of alcohol (70%; 90%; 100%; 100%) and washed in xylene. Then, the slides were cover‐slipped with DPX (Thermo Fisher, Waltham, MA) mounting medium.
Where necessary to confirm the boundaries of the regions of interest, an additional series was mounted onto gelatin‐coated slides and Nissl‐stained using cresyl violet. The sections were then dehydrated through increasing concentrations of alcohol (70%; 90%; 100%; 100%) and washed in xylene. Then, the slides were cover‐slipped with DPX (Thermo Fisher, Waltham, MA) mounting medium.
4
Immunohistochemistry for mCherry Imaging
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Immunohistochemistry was carried out on the tissue to enhance the fluorescence signal of mCherry (DREADD groups). The first series of sections was transferred from PBS into a blocking solution of 5% normal goat serum (NGS) in Phosphate Buffered Saline with Tritonx-1000 (PBST) and incubated for 1 hour. The sections were then transferred into the primary antibody solution of rabbit-anti-mCherry (Abcam, Cambridge, UK) at a dilution of 1:1000 in PBST with 1% NGS and incubated for 24 hours. Sections were washed four times in PBST and transferred to a secondary antibody solution of goat-anti-rabbit (Dylight Alexa flour 594, Vector Laboratories, Peterborough, UK) or at a dilution of 1:200 at PBST. From this point onwards the sections were kept in the dark. Sections were incubated for 1 hour before being washed three times in PBS. Sections were mounted onto gelatin subbed glass slides and were allowed to dry overnight before being immersed in xylene and coverslipped using DPX (Thermo Fisher Scientific, Loughborough, UK). All incubations were on a stirring plate at room temperature and all washes were for 10 minutes. Virus expression was analyzed using a fluorescent Leica DM5000B microscope with a Leica DFC310 FX camera. Images were collected from the anterior cingulate cortex, selected efferent targets, and a comparison cortical area, secondary somatosensory cortex.
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