Five-micrometer sections of formalin-fixed and paraffin-embedded tumor tissue were mounted on charged glass slides and dried at 58 °C for 60 min. Slides were first deparaffinized with xylene then epitope retrieval carried out using citrate buffer in a 2100 Retriever pressure cooker (PickCell Laboratories, San Jose, CA, USA). The sections were blocked with 10% goat serum for 1 h, then incubated with primary antibody (anti-p27, 1 : 50, Abcam (ab7961)) overnight, followed by an HRP-linked goat anti-rabbit secondary antibody (1 : 300, Pierce Biotechnology). The sections were developed with 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA) for 2 min and nuclei counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) for 2 min. PBS washes were performed between all steps. The slides were dehydrated in graded alcohols (100%, 95% and 80% ethanol (vol/vol) in H2O), cleared in xylene and coverslips attached with Permount (Fisher Scientific, Waltham, MA, USA). Slides were scanned using a ScanScope XT Digital Slide Scanner (Aperio, Vista, CA, USA) and data were analyzed with the positive pixel count algorithm (ImageScope, Aperio).
Ab7961
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab7961 is a recombinant monoclonal antibody that recognizes the SH3 domain of the SH3-containing GRB2-like protein 3 (SH3GL3). The antibody is designed for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Ab3085, by Abcam (2 mentions)
Ripa lysis buffer system, by Santa Cruz Biotechnology (2 mentions)
Sc 13024, by Merck Group (2 mentions)
Biotinylated goat anti rabbit, by Vector Laboratories (2 mentions)
P8825, by Abcam (2 mentions)
Anti cytokeratin, by Merck Group (2 mentions)
Avidin fitc, by Vector Laboratories (2 mentions)
Horse anti mouse texas red conjugated antibody, by Vector Laboratories (2 mentions)
Hematoxylin, by Merck Group (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Vector Laboratories (1 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Hrp linked goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab62364, by Abcam (1 mentions)
Sc33536, by Santa Cruz Biotechnology (1 mentions)
Sc33538, by Santa Cruz Biotechnology (1 mentions)
Sc 10790, by Santa Cruz Biotechnology (1 mentions)
C2562, by Merck Group (1 mentions)
Dm5000 fluorescence microscope, by Leica camera (1 mentions)
Vectashield medium with dapi, by Vector Laboratories (1 mentions)
8 protocols using ab7961
1
Immunohistochemical Quantification of p27 in FFPE Tumors
2
Immunohistochemical Evaluation of Immune Cell Antigens
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To evaluate the expression of immune cell antigens using immunohistochemistry, 3 μm tumor sections of paraffin-embedded, formalin-fixed tissues were deparaffinized in xylene and rehydrated in a series of graded ethanol. Endogenous peroxidase activity was blocked by immersing sections in 3% H2O2 for 5 minutes. The sections were blocked in 10% fetal bovine serum for 10 minutes at room temperature and then incubated with primary antibodies to p27Kip1 (rabbit polyclonal, ab7961, 1:100; Abcam) and p-p27Ser10 [EP233(2)Y] (rabbit polyclonal, ab62364, 1:200; Abcam) for 60 minutes of staining. Immunostaining was carried out using the MaxVision™ HRP-Polymer anti-Rabbit IHC Kit (KIT-5005; Maxim) for 15 minutes according to the manufacturer’s protocol and finally visualized with diaminobenzidine. In addition, sections were then counterstained with hematoxylin. The testis tissue served as the positive control.
3
Immunohistochemical Analysis of Parvovirus and p27Kip1
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The organs examined for histopathology were subsequently submitted to immunohistochemical analysis. A commercial mouse monoclonal anti-parvovirus antibody (clone CPV1-2A1, sc57961, Santa Cruz, Dallas, Texas, USA) [7 ] and a polyclonal rabbit anti-human p27Kip1 antibody (ab7961, Abcam, Cambridge, United Kingdom) [11 (link)] were used as primary antibodies (dilution 1:50, for both). Goat anti-mouse or goat anti-rabbit serum (dilution 1/160, 323-005-024 and 323-005-024, respectively, Jackson ImmunoResearch, West Grove, USA) was used as secondary antibody. Binding was visualized using the peroxidase anti-peroxidase method with diaminobenzidine as the chromogen; sections were counterstained with Mayer hematoxylin. Cerebellar sections from an FPV-infected kitten with cerebellar hypoplasia were used as a positive control for the anti-parvovirus immunohistochemistry [7 , 11 (link)], and sections from a fetal cerebellum (estimated gestational age, 54 days) were used as a positive control for the anti-p27Kip1 immunohistochemistry [11 (link)].
4
Protein Expression in P4 Hearts
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Hearts of P4 pups were homogenized and protein isolated using the RIPA lysis buffer system (Santa Cruz Biotechnology). Protein concentrations were quantified using the BCA protein assay (ThermoScientific) and all samples were loaded with equal protein onto 10% polyacrylamide gel with 0.1% sodium dodecyl sulfate (SDS). Proteins were then separated by electrophoresis and transferred onto nitrocellulose membranes. Non-specific binding sites were blocked with Tris-buffered saline solution (TBS) containing 5% dry milk. The membranes were incubated with primary antibodies against cyclin D2 (ab3085, Abcam; 1:1000), and p27 (ab7961, Abcam; 1:1000). After washing, membranes were incubated with secondary antibodies. Proteins were visualized with enhanced chemiluminescence reagents and Western blots were exposed to Hyperfilm. To assure equal loading and minimize variability among gels, samples were normalized to GAPDH.
5
Cardiac Protein Abundance in Anoxia
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HIF-1α, ETA-receptor (ETAR), and ETB-receptor (ETBR) protein abundance in the P4 heart was measured from control and anoxia groups. The protein abundance of cyclin D2 and p27 was measured in P4 hearts from control and anoxia groups as well as in the presence and absence of PD156707. Tissues were homogenized and protein isolated using the RIPA lysis buffer system (Santa Cruz Biotechnology). Protein concentrations were quantified using the BCA protein assay (ThermoScientific) and all samples were loaded with equal protein onto 7.5% (HIF-1α) or 10% (ETAR, and ETBR, cyclin D2, and p27) polyacrylamide gel with 0.1% sodium dodecyl sulfate (SDS). Proteins were then separated by electrophoresis and transferred onto nitrocellulose membranes. Non-specific binding sites were blocked with Tris-buffered saline solution (TBS) containing 5% dry milk. The membranes were incubated with primary antibodies against HIF-1α (sc10790, Santa Cruz Biotechnology; 1:500), ETAR (sc33536, Santa Cruz Biotechnology; 1:500), ETBR (sc33538, Santa Cruz Biotechnology; 1:500), cyclin D2 (ab3085, Abcam; 1:1000), and p27 (ab7961, Abcam; 1:1000). After washing, membranes were incubated with secondary antibodies. Proteins were visualized with enhanced chemiluminescence reagents and western blots were exposed to Hyper film. Kodak image software was used to quantify all results.
6
Immunohistochemical Analysis of Kidney Sections
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Five micron-thick kidney sections were de-paraffinized with xylene and hydrated with graded ethanols. The antigen unmasking and labeling was performed as previously described (Sharma et al., 2005 (link)). Briefly, kidney sections were incubated with 1 M ammonium chloride for 30 min to quench autofluorescence, followed by washing in PBST, and then blocked in 10% normal goat serum (NGS) or 10% normal horse serum (NHS) for 1 h at room temperature. Sections were treated with rabbit anti-Cux1 (1:50, Santa Cruz, sc-13024), mouse anti-PCNA (1:3000, Sigma, P8825), or rabbit anti-p27 (1:100, AbCam, ab7961) primary antibodies overnight at 4 °C. Biotinylated goat anti-rabbit (1:400, Vector) secondary antibody was used to detect Cux1 and p27. The sections were then subsequently probed with FITC-avidin (5 μg/ml, Vector). PCNA antibodies were detected using a horse anti-mouse Texas Red conjugated antibody (Vector, 1:400). To identify collecting ducts, kidney sections were labeled with anti-cytokeratin (1:400, Sigma, C2562). Sections were washed in PBST after the antibody treatments, mounted with Vectashield medium with DAPI (Vector) and slides were viewed on a Leica DM5000 fluorescence microscope and images captured with a Leica DFC365 digital camera.
7
Kidney Immunohistochemistry Protocol
8
Immunohistochemical Analysis of Lung Tissue
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Left lung samples (4 µm) were washed in water for 15 min following dewaxing (xylene for 5 min; xylene and ethanol (1:5) for 5 min; xylene and ethanol (1:1) for 5 min) and incubated with 3% H2O2 for 10 min all at room temperature. Following the antigen was repaired at 95°C for 20 min and the slices were blocked by 0.1% goat serum (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at room temperature for 15 min. Sections were subsequently incubated with primary antibodies against Ki67 (ab15580, 1:100), p27Kip1 (ab7961, 1:100) and cyclin B1 (ab2949, 1:100; all provided by Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with second antibody (1% biotin-labeled goat anti-mouse IgG; ab47844; Abcam) at 37°C for 15 min. This was followed by incubation with horseradish peroxidase-labeled streptomyces ovalbumin at 37°C for 15 min and staining with diaminobenzidine (DAB) for 10–30 min at room temperature. The immunohistochemical results were analyzed using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and semi-quantified to obtain integrated optical density (IOD) values. For each animal, 20 random visual fields of view were observed using an optical microscope (magnification, ×400) and an average IOD value calculated.
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