Mouse brains were freshly dissected and fixed in 4% PFA overnight at 4°C. The frozen tissue sections were prepared as previously described (91 (link)). Primary cultured neurons on a Lab-Tek chamber slides were fixed in 4% PFA for 30 minutes at 37°C. After blocking (5% goat or donkey serum [Sigma-Aldrich], 0.3% Triton X-100 in PBS [pH 7.4]), brain slices or cultured neurons were incubated with primary antibodies against GHSR1a (Santa Cruz Biotechnology, sc-10359, 1:100), DRD1 (Abcam, ab81296, 1:200), PSD95 (Cell Signaling Technology [CST], 3450, 1:400), VGLUT1 (Synaptic Systems, 135304, 1:400), MAP2 (Sigma-Aldrich, M4403, 1:300), DCX (Santa Cruz Biotechnology, sc271390, 1:100), and c-Fos (Synaptic Systems, 226308, 1:400) in mixture or separately. After washing with PBS, the slices or neurons were probed with appropriate cross-adsorbed secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 594, or Alexa Fluor 647 (Thermo Fisher Scientific, 1:500). Images were collected on a Nikon Ti2 confocal microscope. Mean intensity or volume of different staining were analyzed using Nikon-Elements Advanced Research software accordingly.
C fos
Manufactured by Synaptic Systems
Sourced in United States
C-Fos is a protein that is commonly used as a marker for neuronal activity in the central nervous system. It is a transcription factor that is rapidly and transiently expressed in response to various stimuli, making it a useful tool for identifying activated neurons in the brain.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Goat or donkey serum, by Merck Group (1 mentions)
Psd95, by Cell Signaling Technology (1 mentions)
Ghsr1a, by Santa Cruz Biotechnology (1 mentions)
M4403, by Santa Cruz Biotechnology (1 mentions)
Ti2 confocal microscope, by Nikon (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Vglut1, by Synaptic Systems (1 mentions)
H 1400, by Vector Laboratories (1 mentions)
Calbindin, by Abcam (1 mentions)
Alexa fluor 546 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Calbindin d 28k, by Swant (1 mentions)
Eclipse ti2 u, by Nikon (1 mentions)
4 protocols using c fos
1
Immunofluorescence Staining of Mouse Brain
2
Immunohistochemical Labeling of Neuronal Markers
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Mice were deeply anaesthetized with Avertin (350 mg/kg) and transcardially perfused with PBS and 4% PFA sequentially. Brains were postfixed overnight, cryoprotected in 30% sucrose, and cut with a frozen microtome in coronal 50-μm sections. Brain sections were translocated into 24-well plates filled with 1 ml blocking buffer (5% goat serum, 0.3% Triton™ X-100 [Triton was not added when staining membrane proteins, such as TrkB] in 0.1M PBS) for 1 h in the orbital shaker (80 r/min). The 24-well plates were incubated at 4 °C in the orbital shaker (60 r/min) after adding primary antibody. Brain slices in the plate were washed by PBS three times and incubated with secondary antibody at 4 °C overnight. Brain slices were incubated in Hoechst (5 μg/ml) for 10 min at room temperature, washed by PBS three times, and then mounted in mounting medium (H-1400, Vector Laboratories, Inc.) for confocal imaging (Zeiss LSM780 confocal microscope). Primary antibodies for immunostaining were GFP (chicken, 1:100, AVES), TrkB (mouse, 1:50, Santa Cruz), mRFP (rabbit, 1:1000, Takara), orexin (mouse, 1:200, Santa Cruz), MCH (mouse, 1:200, Santa Cruz), and cFos (rabbit, 1:1000, Synaptic Systems).
3
Neuronal Activation Mapping in Mouse Brain
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Mice were sacrificed 90 min after the last trial of the behavioural tasks by a lethal dose of sodium pentobarbital. The brains were removed after transcardial perfusion, and then post-fixed overnight using 4% paraformaldehyde (PFA), after which they were put into 25–30% sucrose solution for 3 days in PBS and then sliced in 30 µm thick coronal sections. For immunofluorescence, sections were washed in PBS-T (PBS consists of 0.1% Triton X-100) and subsequently incubated using Calbindin (1:500; Cat no. Ab108404, Abcam), Calbindin D-28k (1:500; Code No:300, Swant) and/or c-Fos (1:300; Cat no. 226003, Synaptic Systems) for 17–20 h. Followed by 1 h incubation with the secondary antibody using Alexa Fluor 488 donkey to rabbit IgG(H+L), 1:300, Invitrogen A21206; using Alexa Fluor 488 donkey to mouse IgG(H+L), 1:300; Invitrogen A21202 or Alexa Fluor 546 Donkey Anti-Rabbit IgG, 1:300, Invitrogen A10040 at 37 °C, the sections were washed with PBS-T (10 min, three times). Finally, the slices were counterstained with DAPI. Fluorescence images were analyzed by Image J 64. Information of antibodies for immunostaining was listed in Supplementary Table 2 .
4
Immunofluorescence Imaging of Neuronal Activation
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Immunofluorescence images were collected from the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences and Shanghai Key Laboratory of Psychotic Disorders. c-Fos is a marker of neuronal activation which is widely used to locate external stimulation in rodent animals (6 (link), 35 (link)), and c-Fos images obtained by ultrasound stimulation were selected as the training data. Mice were stimulated by ultrasound for 30 min and placed in a quiet room for 60–90 min. Then, the mice were sacrificed and brain tissues were obtained. The brain tissues were cut into slices of a 20 mm thickness and pre-rinsed three times with phosphate buffer saline. The slices were permeabilized and blocked at room temperature for 1 h and incubated with primary antibodies (c-Fos, Synaptic Systems, 226003) at 4°C for over 12 h. The slices were then washed thrice and incubated with secondary antibodies (488 donkey anti-rabbit, ThermoFisher, Waltham, MA, USA, A21206) for 3 h. After washing, the slices were counterstained by 4’, 6-diamidino-2-phenylindole (DAPI). Images were acquired using a Nikon confocal microscope (ECLIPSE Ti2-U, Nikon, Japan).
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