Double-fluorescence staining of CD147 and MMP-11 were conducted on formalin-fixed, paraffin-embedded tissue sections from 20 CRC patients. The slides were baked at 70 °C, dewaxed with xylene, and rehydrated with graded alcohol washes. Antigen retrieval was performed in a pressure cooker, followed by the treatment with 3 % hydrogen peroxide for 15 min to block endogenous peroxidase activity. Thereafter, the sections were incubated at 4 °C overnight with anti-CD147 (ab78106, abcam) or anti-MMP-11(ab52904, abcam). Then the CD147 and MMP-11 primary antibodies were detected by secondary antibodies as follows: Goat anti-mouse IgG (Zhong shan Biotechnology Inc., Beijing, China, ZF-0312, green) and Goat anti-rabbit IgG (Zhong shan Biotechnology Inc., Beijing, China, ZF-0316, red). After washing for two times, nuclei were stained with 4′,6′- diamidino-2-phenylindole (DAPI, Zhong shan Biotechnology Inc., Beijing, China, ZLI-9557) at room temperature for 10 min, and stored at 4 °C. The slides were examined using a laser scanning confocal microscope (Zeiss LSM510). Images were collected and processed using the Zeiss AIM software and sized in Adobe Photoshop CS6.
Goat anti rabbit igg
Manufactured by Zhongshan Biotechnology
Sourced in China
Goat anti-rabbit IgG is a laboratory reagent used in immunological techniques, such as Western blotting and ELISA, to detect the presence of rabbit immunoglobulin G (IgG) in a sample. It is a secondary antibody that binds to rabbit IgG, allowing for the visualization and quantification of target proteins or antigens.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (1 mentions)
Ab78106, by Abcam (1 mentions)
Ab52904, by Abcam (1 mentions)
Goat anti mouse igg, by Zhongshan Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Zhongshan Biotechnology (1 mentions)
Rabbit monoclonal anti vimentin, by Abcam (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti α sma, by Abcam (1 mentions)
Alexa fluor 594 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Ecl kit, by Proteintech (1 mentions)
Ripa lysate buffer, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
3 protocols using goat anti rabbit igg
1
Dual Immunofluorescence Staining of CD147 and MMP-11 in CRC
2
Immunohistochemical Profiling of Cell Markers
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The primary antibodies used were as follows: mouse monoclonal anti-α-SMA (clone: 1A4; dilution: 1:200), rabbit monoclonal anti-tenascin-C (clone: EPR4219; dilution: 1:200), mouse monoclonal anti-cytokeratin (clone: M20; dilution: 1:200), rabbit monoclonal anti-vimentin (clone: EPR3776; dilution: 1:200), and rabbit monoclonal anti-desmin (clone: Y66; dilution: 1:200), all from Abcam (Cambridge, UK). For immunochemical staining, biotinylated horse anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG (dilution: 1:100; Zhongshan Biotechnology, Beijing, China) were used as secondary antibodies. The reaction products were visualized in brown using a diaminobenzidine substrate kit (DAB; Zhongshan Biotechnology, Beijing, China). Counterstaining was performed with haematoxylin or methyl green. For immunofluorescence, Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 594 donkey anti-mouse IgG (dilution: 1:500; Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. diamidino-phenyl-indole (DAPI; dilution: 1:500; Invitrogen, Carlsbad, CA, USA) was used to stain the nuclei. Control staining was performed by substituting non-immune serum (or phosphate-buffered saline) for the primary antibodies. Fluorescence images were acquired using an inverted fluorescence microscope (IX71; Olympus, Tokyo, Japan).
3
Autophagic Activity Quantification in Lung Tissue
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In brief, lung tissue in each group was homogenized in ice-cold RIPA lysate buffer (Sigma) and centrifuged at 15000 ×g for 15 min at 4°C. The total protein concentration was determined with a UV 3000 ultraviolet spectrophotometer (Nano Drop, Wilmington, DE). The samples (100 ug protein per lane) were separated with 15% gradient SDS-PAGE gel and then transferred onto a PVDF membrane. Nonspecific binding to the membrane was blocked with 5% nonfat milk in Tris buffered saline-Tween 20 (TBST, pH 7.4) for 2 h at room temperature. After that the membranes were incubated overnight with rabbit polyclonal antibody against LC3 (1 : 1000 dilution, Cell signaling technology) and β-actin (1 : 1000 dilution, Sigma). After washed repeatedly with TBST, the membrane were incubated in goat anti-rabbit IgG (1 : 5000 dilution, Beijing Zhongshan Biotechnology Co., Ltd., Beijing, China) for 2 h, and bands were visualized by using the ECL kit (Proteintech, USA). The ratio of LC3 level II/LC3I level was used as an indicator of autophagic level.
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