HEK293 cells were transiently transfected with N-terminal Flag-tagged wild type CRF2R or mutants in 24-well plates. 48 hours after transfection, the cells were fixed with 4% (w/v) formaldehyde for 10 min followed by incubation in blocking solution (5% BSA in DPBS) for 1 hour at room temperature. The cells were incubated with anti-FLAG primary antibody (Sigma Aldrich, Cat# F1804, 1:1000) followed by incubation with secondary anti-mouse antibody (Thermo Fisher, Cat# A-21235, 1:5000) conjugated to horseradish peroxide. The tetramethyl benzidine (TMB/E) solution was added and the reaction was terminated by adding 0.25 M HCl solution. The absorbance at 450 nm was measured using the TECAN luminescence counter (Infinite M200 Pro NanoQuant) to characterize the cell surface expression level of each receptor. The expression levels of the mutants were normalized to that of the WT CRF2R. Data are shown as the mean ± SEM. Data are from three independent experiments (n = 3).
Secondary anti mouse antibody
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The secondary anti-mouse antibody is a laboratory reagent used to detect and quantify the presence of mouse primary antibodies in various research and diagnostic applications. It functions by binding to the constant region of mouse primary antibodies, allowing for their identification and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag primary antibody, by Merck Group (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Tmb substrate, by Merck Group (1 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (1 mentions)
Image lab, by Bio-Rad (1 mentions)
R0010, by Solarbio (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Gapdh, by Proteintech (1 mentions)
P0013, by Beyotime (1 mentions)
Tris buffer, by Vector Laboratories (1 mentions)
Fluoromount, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica (1 mentions)
A 31553, by Thermo Fisher Scientific (1 mentions)
7 protocols using secondary anti mouse antibody
1
Quantifying Cell Surface Expression of CRF2R Mutants
2
MRGPRX1 Receptor Expression Assay
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The MRGPRX1 plasmids transfected cells were seeded at a density of 5 × 104 cells per well into 96-well plates and cultured in a 37 °C incubator for 24 h. Cells were fixed by polyformaldehyde for 10 min and then blocked with 5% (w/v) BSA for at least 1 h at room temperature. The washed ELISA plates were probed with an anti-FLAG antibody (Sigma Aldrich, Catalog # F1804, 1:1000) and then incubated with a secondary anti-mouse antibody (Thermo Fisher, Cat# 31430, 1:5000), supplementing with 100 μl TMB substrate (Millipore) until color turned blue. The reaction was stopped with an equal amount of 0.25 M HCl and analyzed by a microplate reader at 450 nm wavelength. The expression levels of the wild-type MRGPRX1 or selective mutants were iteratively adjusted by the quantity of transfected plasmids to assure equal receptor expression in the BRET assay.
3
Protein Expression and Analysis Techniques
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SDS-PAGE, Coomassie staining, and Western blots were performed as described (34) . Unless otherwise described, bacteria equivalent to an OD600 of 0.08 were loaded per lane. Equal loading was validated by 2,2,2-trichloroethane (TCE) staining of total protein (35) . Epitopetagged 3xFLAG proteins were detected using primary antibody anti-FLAG M2 from mouse (diluted 1:4,000, catalogue number F3165; Sigma Aldrich, Germany) and Alexa Fluor 680 fluorescent dye-labeled secondary anti-mouse antibody from goat (diluted 1:10,000, catalogue number A21057; Thermo Scientific, Germany). Quantification of protein bands was performed with Odyssey V3.0 software for Western blots and with ImageLab (BioRad, Germany) for Coomassie stained gels.
U65 and derivatives U65 T1241 ∆(araC-BAD) ∆lac(I-ZYA)FRT Pcp8
U65 ∆pdeLFRT mukB-3xFLAGFRT U467 x pCP20 a Strains were constructed by transduction, which is stated as "x phage[donor strain]"; λ Red recombineering, stated as "x PCR primer pair (template)," followed by Flp recombinase-catalyzed deletion of the resistance marker, "x pCP20". araC Para cra (T7gene10-RBS) in pBAD30 cra (PCR OB161/OB162) in pKECY98 a Features include Cm r (chloramphenicol resistance), Kan r (kanamycin resistance), MCS (multiple-cloning site), and pSC-rep ts (temperature-sensitive replication, derivative of pSC101). b Cloning was verified by sequencing of the cloned PCR fragments.
U65 and derivatives U65 T1241 ∆(araC-BAD) ∆lac(I-ZYA)FRT Pcp8
U65 ∆pdeLFRT mukB-3xFLAGFRT U467 x pCP20 a Strains were constructed by transduction, which is stated as "x phage[donor strain]"; λ Red recombineering, stated as "x PCR primer pair (template)," followed by Flp recombinase-catalyzed deletion of the resistance marker, "x pCP20". araC Para cra (T7gene10-RBS) in pBAD30 cra (PCR OB161/OB162) in pKECY98 a Features include Cm r (chloramphenicol resistance), Kan r (kanamycin resistance), MCS (multiple-cloning site), and pSC-rep ts (temperature-sensitive replication, derivative of pSC101). b Cloning was verified by sequencing of the cloned PCR fragments.
4
Western Blot Analysis of CSF1 in Spinal Cord
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The spinal cord (L4-L6 segments) was dissected and homogenized in ice-cold lysis buffer (P0013, Beyotime) containing protease inhibitor cocktail (R0010, Solarbio). Tissue lysates were centrifuged at 12,000 × g for 15 minutes at 4 °C, and the supernatants were collected for subsequent analysis. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime). Protein samples (30–50 μg per lane) were separated using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (IPVH00010; Millipore). Membranes were blocked for 1 hour in Tris-buffered saline containing 0.1% Tween-20 (TBS-T), followed by overnight incubation at 4°C with the primary mouse antibodies against CSF1 (Santa Cruz Biotechnology) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Proteintech). After washing with TBS-T, the membranes were incubated with secondary antimouse antibody (Invitrogen) for 1 hour. Immunoreactive proteins were visualized with enhanced chemiluminescence (Biosharp), and images were acquired using a luminescent image analyzer (a ChemiDoc MP Imaging System, Bio-Rad). All images were analyzed by Image J (NIH). The results were normalized to GAPDH.
5
Immunohistochemical Analysis of MBP
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The sections obtained from the right hemispheres of animals from groups E and L were pre-incubated in 10% normal goat serum in Tris-Buffer (Vector Laboratories) for 1 h. This step was followed by overnight incubation in mouse primary anti-MBP antibody (1:250, Novocastra), in a humidified chamber at 4 °C. Sections were then washed with phosphate buffer 0.1 M (pH 7.4) and incubated in the secondary anti-mouse antibody (1:400, Invitrogen, EUA) for 2 h at room temperature, followed by DAPI staining for 20 min, then washed with phosphate buffer 0.1 M (pH 7.4) and finally mounted onto histological glass slides with Fluoromount (Invitrogen) to decrease photobleaching. Images of selected sections were acquired with a confocal microscope (Leica TCS SP5), the fluorescence intensity was measured, and Student’s t-test was used to compare sections with and without the primary antibody in experimental and controls animals.
6
Immunofluorescence Staining of Lamin C
7
Immunofluorescence Imaging of Erk5 in Heart
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Sixteen micrometer thick cryosections of hearts were fixed with 4% paraformaldehyde, followed by blocking and permeabilization with 10% normal goat serum and 0.1% Triton-X solution. The primary anti-Flag antibody (Cell Signaling, 8146 1:200) and the secondary anti-mouse antibody (Invitrogen, 1:500) conjugated to Alexa Fluoro568 (Invitrogen) were used to detect the expression of Erk5, whereas DAPI was for nuclear visualization. Images were captured using an Olympus fluorescence microscope.
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