Ninety minutes after retrieval, mice were deeply anesthetized with ketamine/xylazine and intracardially perfused with 0.1 M phosphate buffer (PB) followed by 4% paraformaldehyde (PFA 4%) dissolved in 0.1 M PB. Brains were extracted and post-fixed in PFA 4% for 24 hr. Brains were transferred to 30% sucrose for 48–72 hours before slicing 40µm (for hM4Di-mCherry localization and Nissl staining) or 25 µm (for all immunofluorescence) coronal sections of the entire brain using a cryostat. Sections were stored in phosphate-buffered saline (PBS) with 0.025% sodium-azide at 4°C until use. For immunofluoroscent staining, sections were blocked for 1 hr at room temperature in PBS-T (PBS with 0.25 % Triton X-100) with 8% normal goat serum. Sections were incubated in rabbit anti-Zif268 (Santa-Cruz sc-189; polyclonal; 1:3,000) with mouse anti-PV (Millipore MAB1572; monoclonal; 1:2,000), or rabbit anti-RFP (Rockland 600-401-379; polyclonal; 1:1500) at 4°C for 48–72 hours. Secondary antibodies (Jackson ImmunoResearch; goat anti-rabbit 549 1/1,500, goat anti-mouse 647 1/500) were diluted in the blocking solution and were then applied to the sections for 2 hours at room temperature followed by three rinses for 15 min in PBS-T. Sections were mounted on slides and coverslipped after a brief wash with 0.00005% DAPI in PBS-T to label cell nuclei and stored at 4°C.
Goat anti rabbit 549
Manufactured by Jackson ImmunoResearch
Goat anti-rabbit 549 is a secondary antibody used for the detection and visualization of rabbit primary antibodies in various immunoassays and research applications. This product is conjugated with the fluorescent dye Alexa Fluor 549, which allows for sensitive and specific detection of target proteins.
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Lab products found in correlation
3 protocols using goat anti rabbit 549
1
Immunohistochemistry of Mouse Brain
2
Immunohistochemistry of Mouse Brain
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Ninety minutes after retrieval, mice were deeply anesthetized with ketamine/xylazine and intracardially perfused with 0.1 M phosphate buffer (PB) followed by 4% paraformaldehyde (PFA 4%) dissolved in 0.1 M PB. Brains were extracted and post-fixed in PFA 4% for 24 hr. Brains were transferred to 30% sucrose for 48–72 hours before slicing 40µm (for hM4Di-mCherry localization and Nissl staining) or 25 µm (for all immunofluorescence) coronal sections of the entire brain using a cryostat. Sections were stored in phosphate-buffered saline (PBS) with 0.025% sodium-azide at 4°C until use. For immunofluoroscent staining, sections were blocked for 1 hr at room temperature in PBS-T (PBS with 0.25 % Triton X-100) with 8% normal goat serum. Sections were incubated in rabbit anti-Zif268 (Santa-Cruz sc-189; polyclonal; 1:3,000) with mouse anti-PV (Millipore MAB1572; monoclonal; 1:2,000), or rabbit anti-RFP (Rockland 600-401-379; polyclonal; 1:1500) at 4°C for 48–72 hours. Secondary antibodies (Jackson ImmunoResearch; goat anti-rabbit 549 1/1,500, goat anti-mouse 647 1/500) were diluted in the blocking solution and were then applied to the sections for 2 hours at room temperature followed by three rinses for 15 min in PBS-T. Sections were mounted on slides and coverslipped after a brief wash with 0.00005% DAPI in PBS-T to label cell nuclei and stored at 4°C.
3
Immunolabeling of Neuronal Activation and Synaptic Markers
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Mice were anesthetized and transcardially perfused with ice cold 4% paraformaldehyde 80 min after the start of the retrieval session. Home cage mice, which did not undergo a retrieval session, were perfused on the same day, staggered between perfusions of fear conditioned mice throughout the day. Brains were dissected and postfixed overnight in 4% paraformaldehyde, then sunk in 30% sucrose for 3 d. Brains were sliced into 20-μm coronal sections on a cryostat. Free-floating tissue sections were rinsed three times for 15 min in PBS with 0.25% Triton X-100 (PBS-T), then transferred to a blocking solution of PBS-T with 10% normal goat serum for 1 h at room temperature. Sections were incubated in a primary antibody solution of rabbit anti-Zif268 (Santa Cruz, polyclonal; 1:3000), or rabbit anti-SynapsinI (ThermoScientific; polyclonal; 1:1000) combined with mouse anti-PSD95 (Pierce Antibodies; monoclonal; 1:500). Primary antibodies were diluted in the blocking solution, incubated at 4°C for 72 h, and rinsed three times for 15 min in PBS-T. Secondary antibodies (Jackson ImmunoResearch; goat anti-rabbit 549, 1:1500, goat anti-mouse 647, 1:500) were diluted in the blocking solution and applied to the sections for 2 h at room temperature. Sections were mounted on slides and cover-slipped using DAPI mounting media to label cell nuclei and stored at 4°C.
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