p21 was analyzed by immunohistochemistry. After deparaffinization in xylene and rehydration in graded ethanol, the kidney sections were immersed in proteinase K (Dako, Glostrup, Denmark) for 10 min. The slides were treated with 0.3% hydrogen peroxide for 15 min to block endogenous peroxide activity, and rinsed briefly in PBS. The sections were incubated for 15 min at room temperature with 10% normal goat serum. Then, the sections were incubated one hour at room temperature with a primary anti-p21 antibody (1:100; ab2961; Abcam, Cambridge, UK) and reacted with Histofine Simple-Stain MAX-PO (Nichirei, Tokyo, Japan) for 30 min at room temperature. The sections were then developed with diaminobenzidine using Simple stain DAB buffer (Nichirei). After the immunohistochemistry, the slides were stained with hematoxylin to stain the nucleus or periodic acid-Schiff (PAS) reagent to stain the brush border in the proximal tubules for analyzing the location of p21-positive tubular cells. The number of p21-positive cells (defined as those stained brown) in the renal cortical area was counted in five randomly chosen fields per section at a magnification of 200× by using hematoxylin-stained sections.
Ab2961
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab2961 is a mouse monoclonal antibody that recognizes the protein Vimentin. Vimentin is a type III intermediate filament protein expressed in various cell types. This antibody can be used for the detection of Vimentin in immunohistochemistry and Western blot applications.
Automatically generated - may contain errors
Lab products found in correlation
Histofine simple stain max po, by Nichirei Biosciences (1 mentions)
Proteinase k, by Agilent Technologies (1 mentions)
α sma, by Abcam (1 mentions)
Pa1 822a, by Thermo Fisher Scientific (1 mentions)
U lh100 3, by Olympus (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail solution, by GenDEPOT (1 mentions)
Anti hp1γ, by Abcam (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti rabbit igg hrp, by Merck Group (1 mentions)
Ecl western blot detection system, by Cytiva (1 mentions)
Anti phospho mek, by Cell Signaling Technology (1 mentions)
Anti p27 kip1, by Abcam (1 mentions)
Anti p16ink4a 10883 1 ap, by Proteintech (1 mentions)
Anti pan ras, by Santa Cruz Biotechnology (1 mentions)
Anti dcr 2, by Abcam (1 mentions)
3 protocols using ab2961
1
Immunohistochemical Analysis of p21 in Kidney
2
Immunofluorescence Staining of PPARα and αSMA
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The frozen sections were permeabilized with 0.1% Triton X-100 (9002-93-1, Sigma, MA, UK) and blocked with 10% goat serum (ZLI-9021, ORIGINE, Beijing, China) for 30 min at room temperature. The sections were incubated with the following diluted primary antibodies overnight at 4 °C: PPARα (1:100 dilution, PA1-822A, Invitrogen, Waltham, MA, USA) and αSMA (1:200 dilution, ab2961, Abcam, Madison, WI, USA). The fluorescent secondary antibodies, goat anti-rabbit TRITC (ZF-0316, ORIGINE, Beijing, China) and goat anti-mouse FITC (ZF-0312, ORIGINE, Beijing, China), were mixed and added, and the slides were further incubated at 37 °C for 1 h. The sections were then incubated with 4′,6′-diamidino-2-phenylindole (DAPI) and mounted. The images were acquired using an Olympus laser scanning microscope (U-LH100-3, Olympus Corporation, TKY, JPN).
3
Profiling RAS/MAPK Signaling in Liver
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Liver tissues were homogenized and digested in 1× RIPA buffer containing phosphatase inhibitor cocktail solution (GenDEPOT, Barker, TX, USA). Western blot experiments were performed following the standard protocol. The following primary antibodies were used: anti-Pan-RAS (sc-14022; Santa Cruz Biotechnology), anti-phospho-AKT (#4060, Cell Signaling Technology), anti-phospho-MEK (#9154, Cell Signaling Technology), anti-phospho-ERK (#4370, Cell Signaling Technology), anti-p21Cip1 (ab2961; Abcam), anti-p27Kip1 (ab7961; Abcam), anti-p16INK4A (10883-1-AP; Proteintech), anti-HP1γ (ab10480, Abcam), anti-DcR2 (ab2019; Abcam), and anti-GAPDH (#2118; Cell Signaling Technology). Anti-rabbit IgG–HRP (Sigma-Aldrich) was used as the secondary antibody. Bands were detected using the enhanced chemiluminescence (ECL) Western blot detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
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