Epithelial ovarian cancer tissue was cut into 4-micron-thick serial sections, fixed in formalin and embedded in paraffin. These paraffin sections were baked at 65°C for 30 min. To deparaffinize and rehydrate, the slides were placed in hydrogen peroxide (concentration 3%). All slides were immersed in 0.01 mol/l EDTA, autoclaved at 121°C for 4 min, cooled to room temperature, immersed three times with distilled water for 2 min, and washed three times with PBS for 5 min. Anti-RRBP1 antibody (Abcam, Anti-RRBP1 antibody ab95983) was used overnight at 4°C at a 1:400 dilution. The antibody was then immobilized three times with PBS for 5 min, placed into the moisture box, supplemented with the secondary antibody, and then placed in the humidor for 20 min at room temperature. The slides were counterstained with Hematoxylin, dehydrated, sealed, and placed in an oven at 60°C for 48 h.
Ab95983
Manufactured by Abcam
Ab95983 is a laboratory equipment product. It serves as a core function without further interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Ab22595, by Abcam (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Ab32374, by Abcam (1 mentions)
Amicon ultra 15 tube, by Merck Group (1 mentions)
Hpa011924, by Merck Group (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Immobilon p transfer polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Mab374, by Merck Group (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
Ht501128, by Merck Group (1 mentions)
Pab28477, by Abnova (1 mentions)
A21428, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
4 protocols using ab95983
1
Immunohistochemical Analysis of RRBP1 in Ovarian Cancer
2
Protein Extraction and Western Blot Analysis
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Mouse tissues and cell lysates were extracted using RIPA lysis buffer (20–188, Millipore) according to the manufacturer’s protocol. Cell culture supernatant was collected and concentrated with Amicon Ultra-15 tubes (10 KDa, Millipore). The following antibodies were used: anti-RRBP1 (PA5-21,392, Invitrogen), anti-RRBP1 (Ab95983, Abcam), anti-RRBP1 (HPA011924, Sigma), anti-Hsp70 (DF2698, Affinity), anti-β-actin (GTX109697, Genetex), anti-renin (H0005972-M01, Abnova), anti-ACE (MA5-32,741, Invitrogen), anti-β-tubulin (tcaba2, Taiclone), anti-SGK1 (ab32374, Abcam), anti-ADCY5/6 (PA5-75,274, Abnova), and anti-calnexin (ab22595, Abcam). The blots were detected with Trident pico Western HRP Substrate (GTX17435, Genetex), and images were analyzed using a UVP BioSpectrum Imaging System.
3
Quantitative Western Blot Analysis
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Cell lysates were extracted using RIPA buffer and quantified using a BCA protein analysis kit (Pierce, Rockford, IL, USA). Twenty micrograms of lysate was loaded onto a polyacrylamide gel and SDS-PAGE was performed. The separated proteins were transferred to an Immobilon-P transfer polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Each membrane was incubated with primary antibody to RRBP1 (ab95983; 1:1000 dilution; Abcam) and glyceraldehyde 3-phosphate dehydrogenase (MAB374; 1:1000 dilution; Millipore) overnight and for 2 h, respectively. After reaction with horseradish peroxidase-conjugated secondary antibody (1:2000 dilution; Cell Signaling Technology, Beverly, MA, USA) for 1 h, each membrane was scanned using a UVP ChemStudio PLUS instrument (UVP Inc., Upland, CA, USA) and analyzed with the ImageJ software (version 1.8).
4
Immunofluorescence Microscopy of Organelle Markers
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Cells were cultured on coverslips and then fixed in 10% formaldehyde solution (HT501128, Sigma) for 10 min at room temperature, followed by washing thrice with PBS for five minutes each. The cells were incubated overnight at 4 °C with the following primary antibodies in blocking buffer: mouse anti-renin (1:50, H00005972-M01, Abnova), rabbit anti-calnexin (1:400, ab22595, Abcam), rabbit anti-GOLIM4 (1:200, PAB28477, Abnova), and rabbit anti-RRBP1 (1:200, ab95983, Abcam) followed by Alexa Fluor® 488 goat anti-mouse IgG(H+L) (1:100, A11029, Thermo Fisher) or Alexa Fluor™ 555 goat anti-rabbit IgG(H+L) (1:100, A21428, Thermo Fisher) secondary antibody for 1.5 h at room temperature. Finally, slides were mounted with a drop of DAPI Fluoromount-G® (0100–20, Southern Biotech). Samples were examined using a laser scanning confocal microscope (Zeiss LSM 700) and imaged with a 63 × oil objective lens in a 1024 × 1024 pixel format at a 12-bit intensity resolution. Additional file 1 details the in vitro analysis.
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