Spinal cord tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 4 μm. After the deparaffinization and rehydration, the spinal cord sections were immersed in citrate antigen retrieval solution (Beyotime, China) for antigen repair, incubated with 3% hydrogen peroxide for 10 min blocked with Immunol Staining Blocking Buffer (Beyotime, China) for 60 min, incubated with anti-SNAP25 rabbit pAb (A2234, ABclonal), GATM rabbit pAb (1:1000, A6598), and NDUFA11 rabbit pAb (1:1000, A16239) overnight at 4°C and goat anti-rabbit IgG H&L (FITC) (ab6717, USA) at room temperature for 60 min. The images were captured with a fluorescence microscope (Olympus IX73, JPN). The relative fluorescence intensities were analyzed by ImageJ software (Yu et al., 2020 (link)).
Goat anti rabbit igg h l
Manufactured by ABclonal
Sourced in United States
Goat anti-rabbit IgG H&L is a secondary antibody that specifically binds to the heavy and light chains of rabbit immunoglobulin G (IgG) antibodies. It is commonly used in various immunoassays and detection techniques to identify and quantify the presence of rabbit IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Immunol staining blocking buffer, by Beyotime (1 mentions)
Citrate antigen retrieval solution, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Anti gapdh, by ABclonal (1 mentions)
P iκbα, by ABclonal (1 mentions)
Ipvh00005, by Merck Group (1 mentions)
Ap0707, by ABclonal (1 mentions)
A19056, by ABclonal (1 mentions)
3 protocols using goat anti rabbit igg h l
1
Immunofluorescence Analysis of Spinal Cord Tissues
2
Quantification of Cas9 Expression in Doxycycline-Induced HEK293T Cells
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For Western blot, HEK293T clone cells were lysed 5 days after Doxycycline was added using RIPA buffer supplemented with proteinase and phosphatase inhibitors. Total protein was quantified using the BCA kit (Beyotime cat# P0012). 20 µg per well of total protein was separated by electrophoresis using a 15-well 4–12% Tris-Gly and transferred to a PVDF membrane for 120 min at 300 mA before blocking with 10% skimmed milk powder for 2 h at 4 °C. PVDF membranes were incubated with a 1:1000 dilution of anti-GAPDH (ABclonal, A19056) and a 1:1000 dilution of anti-Cas9 (ABclonal, A14997) overnight. Then, membranes were incubated with a 1:1000 dilution of HRP Goat Anti-Rabbit IgG (H + L) (ABclonal, AS014) for 2 h and visualized using Tanon imager (Supplementary Fig. 4b ).
3
Protein Extraction and Western Blot Analysis
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Cellular proteins were isolated using radio-immuno precipitation assay (RIPA) buffer (P0013B, Beyotime, Shanghai, China), followed by separation via 8% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and their transfer onto polyvinylidene fluoride (PVDF) membranes (IPVH00005, Millipore, Billrica, MA, USA). Afterward, the membranes underwent blocking with 5% Bovine Serum Albumin (BSA) for 1 hour, followed by PBS washing. Subsequently, the membranes underwent an overnight incubation at 4 °C with the respective primary antibodies: p-IκBα (AP0707, 1:1000, ABclonal, Wuhan, China), NF-κB (A14754, 1:1000, ABclonal, Wuhan, China), Notch1 (A7636, 1:1000, ABclonal, Wuhan, China), Hes-1 (A0925, 1:1000, ABclonal, Wuhan, China), GAPDH (A19056, 1:5000, ABclonal, Wuhan, China). After washing with PBS, the membranes underwent incubation with the corresponding secondary anti-body, goat anti-rabbit IgG (H+L) (AS014, 1:2000, ABclonal, Wuhan, China), for 2 hours at room temperature. After three washes with tris-buffered saline Tween-20 (TBST) buffer (60145ES76, Yi Sheng Biotechnology, Shanghai, China) for 5 minutes each, signal detection was performed utilizing an enhanced chemiluminescence detection kit (P0018S, Beyotime, Shanghai, China). Image J software (V1.8.0.112, NIH, Madison, WI, USA) was used for image analysis.
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