Brain slices were double-stained for immunohistochemical evaluation using fate-specific antibodies including CRH/BDNF and GR/MR. Briefly, the brain sections were incubated in 3% hydrogen peroxide for 0.5 h and then blocked with normal bovine serum for 0.5 h. The brain sections were washed three times and incubated with rabbit polyclonal anti-CRH (1:1000, ab8901, Abcam, UK), mouse monoclonal anti-BDNF (1:500, ab108319, Abcam, UK), rabbit polyclonal anti-GR (1/200 dilution; ab3578, Abcam, UK), and mouse monoclonal anti-MR (1/200 dilution; ab2774, Abcam, UK) overnight at 4 °C. The slices were then washed with PBS and incubated with Alexa Fluor 647-conjugated donkey anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG at room temperature for 2 h. Finally, the samples were counterstained with DAPI (Sigma-Aldrich, St. Louis, MO) for 10 min. Digital images of the whole brain sections were obtained by a MIDI FL (3D Histech, Hungary) system.
Ab2774
Manufactured by Abcam
Sourced in United Kingdom
Ab2774 is a laboratory equipment product offered by Abcam. It serves as a tool for scientific research and experimentation. The core function of Ab2774 is to facilitate specific tasks or measurements required in a laboratory setting. No further details or interpretations about the intended use of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab3578, by Abcam (2 mentions)
Ab8901, by Abcam (1 mentions)
Midi fl, by 3DHISTECH (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab108319, by Abcam (1 mentions)
Ab6747, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab8245, by Abcam (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Ab33186, by Abcam (1 mentions)
Ab59337, by Abcam (1 mentions)
T7816, by Merck Group (1 mentions)
Ab216476, by Abcam (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Ab196495, by Abcam (1 mentions)
Histone h3, by Merck Group (1 mentions)
Ab179467, by Abcam (1 mentions)
4 protocols using ab2774
1
Dual Immunofluorescence Staining of Brain Slices
2
Quantification of NR3C2 Protein Levels
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Firstly, total proteins were obtained from cells in RIPA buffer (Solarbio Science & Technology Co. Ltd., Beijing, China) containing proteinase inhibitor. Next, a BCA protein assay kit was adopted to quantify protein, SDS-PAGE gels were used to separate protein, and then protein samples were transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA) with 5% skim milk. Subsequently, the membrane was co-incubated with anti-NR3C2 (1:1000, ab2774) and anti-GAPDH (1:1000, ab8245) at 4 °C all night, and then incubated with secondary antibody rabbit anti-sheep IgG H&L (HRP) (1:2000, ab6747) in a dark room at 37 °C for 1 h. Antibodies used in Western blot assay were acquired commercially from Abcam (Cambridge, USA). Finally, the immunoblots were subjected to enhanced chemiluminescence reagent. GAPDH was used as the loading control.
3
Kidney Protein Expression Analysis
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Kidney sample was homogenized in RIPA buffer by smashing in liquid nitrogen. Same amount of lysates protein (40 µg) was loaded and separated in SDS-PAGE. The resolved proteins were transferred onto nitrocellulose membranes and probed with specific primary antibody at 4°C overnight with agitation. GAPDH was probed as a loading control. The protein bands were visualized using enhanced chemiluminescence (ECL) reagents (GE Healthcare, Chicago, Illinois, United State) and developed in Fujitsu Biomedical film (Fujitsu, Japan). Densitometric analysis was performed using the ImageJ (NIH). Quantification protein expression was based on the ratio of target protein to GAPDH. The following primary antibodies were used: anti-ACE (Ab216476, Abcam, 1:500), anti-FGF2 (05-118, Millipore, 1:1000), anti-NHE3 (NB110-61586, Novus Biological, 1:2000), anti-Rac1 (Ab33186, Abcam, 1:1000), anti-Nr3c2 (Ab2774, Abcam, 1:1000, anti-Sgk1 (Ab59337, Abcam, 1:1000), anti-β-tubulin I (T7816, Sigma-Aldrich, 1:30000), and anti-GAPDH (2251-1, Epitomics, 1:30000).
4
Protein Expression Profiling in Rat Hippocampus after CCI
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The rats (n=16 per group) were sacri ced by decapitation on day 3 after CCI and the right hippocampus was removed at 4°C. Total (n=8 per group) and nuclear protein (n=8 per group) extraction were performed as previously described [25] . Equal amounts of proteins (30 μg) were separated by SDS-PAGE and then transferred onto polyvinylidene uoride membranes. Blots were incubated in 5% nonfat milk or BSA for 2 hr and then reacted overnight at 4°C with primary antibody P-Akt (ser 473, 1:5000; ab81283), P-Ert (1:1000, CST, #9010), P-CREB (ser133, 1:5000; ab32096), P53 (1:1000, ab131442), Bax (1:1000, ab32503), P-Bad (S136, 1:1000, ab28824), cleaved caspase-3 (1:1000, #9664, CST), Bcl-2 (1:1000; ab196495), MR (1:400, ab2774), GR (1/200 dilution; ab3578), β-Actin (1:5000; ab179467, Abcam, UK), and Histone H3 (1:400; Millipore Co.), followed by incubation with secondary antibodies for 2 hr at room temperature. Blots were visualized using chemiluminescence (Bio Spectrum 500 Imaging System; UVP Co., Upland, CA, USA) and ImageJ software determined the intensity of each band. The percent expression compared with that of the sham controls was calculated for each sample.
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