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.
Ghsr1a
Manufactured by Santa Cruz Biotechnology
Sourced in United States
GHSR1a is a G protein-coupled receptor that serves as the receptor for the hormone ghrelin. It plays a role in the regulation of growth hormone secretion, appetite, and energy homeostasis.
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Lab products found in correlation
Ti2 confocal microscope, by Nikon (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Goat or donkey serum, by Merck Group (1 mentions)
M4403, by Santa Cruz Biotechnology (1 mentions)
C fos, by Synaptic Systems (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Psd95, by Cell Signaling Technology (1 mentions)
Vglut1, by Synaptic Systems (1 mentions)
Akt and p akt, by Cell Signaling Technology (1 mentions)
Ezrgra 90k, by Merck Group (1 mentions)
Gapdh, by Proteintech (1 mentions)
Anti rabbit plus, by Merck Group (1 mentions)
Anti goat minus, by Merck Group (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
β actin, by MP Biomedicals (1 mentions)
Pparγ, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Merck Group (1 mentions)
5 protocols using ghsr1a
1
Immunofluorescence Staining of Mouse Brain
2
Serum Ghrelin and Lactate Measurement Protocol
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At sacrifice, 2–3 h after lights on, the serum was used for measurement of ghrelin and lactate, using a specific enzyme-linked immunosorbent assay kit15 (link)–20 (link). The low and high detection limits for ghrelin were 0.04 and 10 ng/mL (kit number EZRGRA-90K; Merck Millipore, Rosh Haayin, Israel), and the intra- and inter-assay CVs were 1.1% and 3.2%, respectively. Serum lactate was determined at the Soroka University Medical Center Biochemistry laboratory. In a subset of n = 4 of control animals, AO, and OR arterial blood gases (pH, PCO2, PO2, and HCO3−) were determined in parallel to their serum lactate level on day 66 after surgery. Antibodies used for evaluation of the hypothalamic protein extract by western immunoblot analysis (see Supplementary Methods ) were GHSR1a (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Akt and p-Akt (Cell Signaling Technology, Danvers, MA, USA), soleus AMPK and p-AMPK (MP Biomedical Solon, OH, USA), and GAPDH (Proteintech, Rosemont, IL).
3
GHSR and DRD1 Protein Interaction Detection
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Protein interactions between GHSR/DRD1 in mouse brain slices were detected using Duolink PLA detection kits (Sigma-Aldrich, DUO92008) following manufacturer’s instructions. The following primary antibodies were used: GHS-R1a (Santa Cruz Biotechnology, sc-10359, 1:100) and anti-DRD1 (Abcam, ab81296, 1:200). The specificity of antibodies to GHSR and DRD1 was validated as previously described (17 (link)). The following Duolink in Situ PLA Probes were used: anti–rabbit PLUS (Sigma-Aldrich, DUO92002) and anti–goat MINUS (Sigma-Aldrich, DUO92006). Images were collected on a Nikon Ti2 confocal microscope. The mean intensity of PLA+ signals was analyzed using Nikon-Elements Advanced Research software.
4
Antibody Evaluation of EGP Extracts
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The following antibodies were used for evaluation of the EGP extracts by western immunoblot15 (link),17 (link),29 (link): GHSR1a (n = 6 in each group), Sox9 (n = 8 in each group), PPARγ (n = 6 in each group) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), OX1R (n = 8 in each group) (Abcam, Cambridge, MA, USA), AKT (n = 6 in each group), p-AKT (n = 6 in each group), AMPK (n = 8 in each group), p-AMPK (n = 8 in each group) (Cell Signaling Technology, Danvers, MA, USA), and β-actin (MP Biomedical Solon, OH, USA). The full protocol is described in our previous publication29 (link). Serum TRAP 5b was measured using an ELISA kit (MBS704438; MyBioSource, Inc., San Diego, CA, USA). The low and high detection limits were 0.312 and 40 mlU/ml, and the coefficient of variation was <5%.
5
Western Blot Analysis of GHS-R1a
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Frozen tissue samples from striatum, substantia nigra, frontal cortex and hippocampus of 3 adult female SD rats and 7 VM sections from embryos of three E14 Wistar rats were mixed with lysis buffer solution containing anti-protease and anti-phosphatase solutions. Samples were homogenised in tubes containing ceramic beads using Precellys®24 homogenizer followed by centrifugation to remove undissolved impurities. protein concentration in the supernatant was estimated using Bicinchoninic acid assay kit followed by mixing the protein solution with Laemmli lysis buffer and denaturation at 90 for 5 min. The protein samples were loaded in 10% SDS-PAGE running gel and separated in an electrophoresis chamber at 200 V for 1-3 hrs. Then the protein extract was transferred onto nitrocellulose blotting membrane using a semi-dry transfer method. The membrane was blocked with 5% skimmed milk and left with primary antibody overnight at 4 0 C (GHS-R1a 1:200, Santa Cruz Biotechnology; GAPDH 1:15000, Sigma). The membrane was washed and incubated with secondary antibody for 1hr followed by washing and developing the bands using SuperSignal® West Dura kit.
After that, the bands were visualised using gel imaging Syngene® G BOX linked to an automatic control software (GeneSys).
After that, the bands were visualised using gel imaging Syngene® G BOX linked to an automatic control software (GeneSys).
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