The slides were dried, dewaxed, rehydrated, and treated with 3% hydrogen peroxide (H2O2) in methanol for 10 min. Antigen retrieval was performed by boiling the slides twice in a sodium citrate buffer (0.01 mol/L, pH 6.0). A 5% bovine serum albumin (BSA; Boster Biological Technology Co. Ltd, Wuhan, China) was used in a 1:10 dilution during a 30 min incubation period at 37 °C. There was a two-hour incubation period with the Ki67 antibody (Abcam, ab15580; 1:800 dilution) at 37 °C. Sections were treated with a goat anti-rabbit IgG secondary antibody (ZSGB-BIO, Beijing, China) for one hour at 37 °C. With the exception of the blocking step, at every step, washing was completed thrice in PBS for 5 min. Positive cells were observed with a diaminobenzidine (DAB) Kit (ZSGB-BIO, Beijing, China), stained with hematoxylin, and made into permanent pieces. A total of 15 microscopic fields per sample were captured using a light microscope under 20× magnification (Leica DM3000, Leica Microsystems, Wetzlar, Germany). The number of Ki67-positive cells, at least 30 well-oriented complete crypts per sample was counted manually for analysis [24 (link)].
Goat anti rabbit igg secondary antibody
Manufactured by ZSGB-BIO
Sourced in China
The Goat anti-rabbit IgG secondary antibody is a reagent used in immunological techniques, such as Western blotting, ELISA, and immunohistochemistry. It is designed to bind to and detect rabbit primary antibodies, allowing for the visualization and quantification of target proteins or antigens.
Automatically generated - may contain errors
Lab products found in correlation
Ki67 antibody, by Abcam (1 mentions)
Leica dm3000, by Leica Microsystems (1 mentions)
Diaminobenzidine dab kit, by ZSGB-BIO (1 mentions)
Complete protease inhibitor cocktail, by Beyotime (1 mentions)
Micro bca protein assay kit, by Beyotime (1 mentions)
Rabbit anti β actin monoclonal antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Luminescent image analyzer, by GE Healthcare (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Anti psd 95 antibody, by Abcam (1 mentions)
Gel imager, by Bio-Rad (1 mentions)
Ripa lysis, by Solarbio (1 mentions)
Ecl solution, by Beyotime (1 mentions)
Gapdh, by Bio-Rad (1 mentions)
Dnmt1, by Cell Signaling Technology (1 mentions)
Gapdh, by ZSGB-BIO (1 mentions)
Gel imaging system, by Analytik Jena (1 mentions)
β actin antibody, by ZSGB-BIO (1 mentions)
7 protocols using goat anti rabbit igg secondary antibody
1
Ki67 Immunohistochemistry Protocol
2
Protein Expression Analysis of Burn Wounds
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Mice used for western blot analysis were sacrificed at 0, 6, 12, 24, 48 and 72 h post burns. Total protein from burn wounds were obtained using lysis buffer supplemented with complete protease inhibitor cocktail (Beyotime, China). A micro-BCA protein assay kit (Beyotime, China) was used to measure protein concentration in each sample. Total protein was then separated by s odium d odecyl s ulfate-p olya crylamide g el e lectrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore, Bedford, MA, USA), which was further incubated with rabbit anti-Hsp90α monoclonal antibody (1∶1000 dilution, Epitomic, USA) or rabbit anti-β-actin monoclonal antibody (1∶1000 dilution, Cell Signaling, USA) at 4°C overnight, followed by incubation with goat anti-rabbit IgG secondary antibody (1∶3000, ZSGB-BIO, Beijing, China). Protein bands were visualized by FluorChem FC digital imaging system (Alpha Innotech).
3
Protein Expression Analysis by Immunoblotting
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Total protein was extracted using RIPA buffer with protease inhibitors (Roche). For immunoblot analysis, protein samples were resolved by SDS–PAGE and transferred to nitrocellulose membranes with an iBlot 2 dry blotting system (Thermo Fisher Scientific). Membranes were blocked in 5% nonfat dry milk in TBST (100 mmol/L Tris, pH 7.5, 0.9% NaCl, 0.1% Tween-20) for 1 hour and incubated with primary antibodies overnight at 4 °C (rabbit anti-eNOS, 1:1000, No. ab5589, Abcam; rabbit anti-beta actin, 1:1000, No 20536-1-AP, Proteintech; rabbit anti-PKG, 1:500, No. bs-6705R, Bioss; rabbit anti-sGC, 1:500, No. bs-13544R, Bioss). The membranes were washed with TBST and incubated for 1 h with goat anti-rabbit-IgG secondary antibody (1:8000, ZSGB-Bio) at room temperature. The blots were developed using ECL (Thermo Fisher Scientific), and images were captured with a luminescent image analyzer (GE).
4
PSD95 Expression in Hippocampal Regions
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The mice were perfusion-fixed with PFA, and their brains were removed. The brains were cut from superior colliculus to optic chiasma, conventionally dehydrated, immersed by wax, embedded and sectioned into 5 μm thick slices. After dewaxing and hydration, the sections were subjected to high-pressure antigen repairing. Subsequently, they were blocked and incubated overnight at 4°C with anti-PSD95 antibody (cat#: ab18258, Abcam, United States). Afterward, the sections were incubated with goat anti-rabbit IgG secondary antibody (cat#: SP-9001, ZSGB-BIO, China) for 1 h, horseradish enzyme-labeled streptavidin for 1 h, followed by DAB staining and hematoxylin counterstaining. Images of hippocampal CA1 and CA3 were observed and collected under a 40 × light microscope (Leica, Germany), and the average optical density was analyzed using the Image J software (National Institutes of Health, United States).
5
Protein Expression Analysis in Cells
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Total protein was extracted using RIPA lysis (R0010, Solarbio, Beijing, China), followed by protein concentration measurement with BCA kits (GBCBIO Technologies, Guangzhou, China). Afterwards, the protein (40 µg for each sample) was separated by SDS-PAGE, electro-transferred to PVDF membrane, and blocked with 5% BSA. Subsequently, the immunoblots were probed with diluted primary antibodies against DNMT1 (1:1000, #5032, Cell Signaling Technology [CST], Beverly, MA), BAG3 (1:5000, ab92309, Abcam), HSPB8 (1:1000, #3059, CST), mTOR (1:1000, #2972, CST), p-mTOR (1:1000, #5536, CST), p70 S6 (1:1000, #9202, CST), p-p70 S6 (1:1000, #9234, CST), AKT (1:1000, #9272, CST), p-AKT (ser473) (1:1000, #4060, CST), LC3-II/I (1:200, #4180, CST), ATG-3 (1:1000, #3415, CST), ATG-5 (1 :1000, #2630, CST), ATG-7 (1:1000, #2631, CST), ATG-12 (1:1000, #4180, CST), Beclin (1:1000, #3738, CST) and GAPDH (1:2000, ZSGB-BIO, Beijing, China) overnight at 4°C, followed by 1-h incubation with goat anti-rabbit IgG secondary antibody (1:2000, ZSGB-BIO). Protein bands were then visualized utilizing ECL solution (Beyotime) and Bio-Rad gel imager, followed by quantification by the ImageJ analysis software (normalized to GAPDH).
6
Myocardial Protein Expression Analysis
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The left ventricle wall was collected and rapidly frozen in liquid nitrogen and stored at −80°C. The heart was removed from a −80°C freezer, and the left ventricular myocardial tissue was sheared on ice. The myocardial tissue was homogenized to directly collect the supernatant liquor. The protein sample was mixed with buffer and heated to denaturation, and then SDS-PAGE was added. The sample was transferred to a polyvinylidene fluoride (PVDF) membrane, sealed with a sealing solution for 1 h, and then incubated overnight at 4°C with the corresponding primary antibodies, including p-PI3K antibody (Bioss, China, 1 : 600), p-Akt antibody (Bioworld, China, 1 : 600), Bad antibody (Bioss, China, 1 : 600), Bcl-2 antibody (Abcam, UK, 1 : 500), Bax antibody (Abcam, UK, 1 : 1000), Caspase-3 antibody (Santacruz, USA, 1 : 400), and β-actin antibody (ZSGB-BIO, China, 1 : 1000). The reaction membrane was incubated with secondary goat anti-rabbit IgG antibody (ZSGB-BIO, China, 1 : 3000) at room temperature for 1 h. The ECL luminescence kit (Santacruz, USA) was used for colorimetric detection. Images were scanned and measured in terms of the grayscale values by the gel imaging system (UVP, USA).
7
Immunohistochemical Analysis of UCP1 in Mouse iWAT
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Hematoxylin and eosin (H&E) staining and UCP1 immunohistochemical staining were performed in mouse iWAT according to standard procedures. A primary rabbit polyclonal antibody against UCP1 (1:200 dilution, Abcam, Cambridge, UK) and a secondary goat anti-rabbit IgG antibody (1:2,000 dilution, ZSGB-BIO, Beijing, China) were used in this process. All sections for UCP1 staining were counter-stained with Harris hematoxylin (Histolab, Gothenburg, Sweden) and observed by light microscopy.
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