Total protein from lung tissues and cells was isolated with radioimmunoprecipitation (RIPA) lysis buffer (Beyotime, Jiangsu, China) and quantified using the BCA Protein Quantification Kit (Solarbio, Beijing, China). Then, 20 μg proteins were submitted to electrophoresis on 10% sodium dodecyl sulfatepolyacrylamide gel, and were then transferred into the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After that, the membranes were blocked with 5% non-fat milk and probed with the primary antibodies against ZIC2 (No. ab150404, Abcam, MA, USA), HOXA1 (No. ab230513, Abcam), HOXA11 (No. ab72591, Abcam) or GAPDH (Abcam) at 4° C overnight. Next, the membranes were incubated with the corresponding secondary antibodies for 1 hour at room temperature. The western blots were visualized using the enhanced chemiluminescence reagents (Millipore) and detected on iBright CL1500 Imaging System (Thermo Fisher Scientific, USA), and analyzed by ImageJ software. GAPDH serves as an internal reference.
Ab230513
Manufactured by Abcam
Sourced in United Kingdom
Ab230513 is a primary antibody used in immunoassay applications. It is a purified polyclonal antibody produced in rabbit.
Automatically generated - may contain errors
Lab products found in correlation
Ab72591, by Abcam (1 mentions)
Ab150404, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ibright cl1500 imaging system, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
Bca protein quantification kit, by Solarbio (1 mentions)
Tween 20, by Thermo Fisher Scientific (1 mentions)
Bca protein quantification kit, by Beyotime (1 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by Abcam (1 mentions)
Sds page, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab191470, by Abcam (1 mentions)
Ab229960, by Abcam (1 mentions)
Ab230879, by Abcam (1 mentions)
Bx50 bx fla dp70, by Olympus (1 mentions)
Imagej pro, by Media Cybernetics (1 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
6 protocols using ab230513
1
Quantification and Analysis of Lung Protein Levels
2
Western Blot Analysis of HOXA1 Protein
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Total protein of tissues and cultured cells were extracted using RIPA buffer (Beyotime Institute of Biotechnology), and was quantified by a BCA Protein Quantification Kit. Total protein (20 µg) was separated using 10% sodium dodecyl sulfonate-polyacrylamide gel (SDS-PAGE; Beyotime Institute of Biotechnology). Then the proteins (20 µg/lane) were transferred onto polyvinylidene difluoride membranes (PVDF; EMD Millipore). Membranes were blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) in TBS with 0.1% Tween-20 (Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Next, the membranes were incubated with primary antibodies against HOXA1 (1:500 dilution; product code ab230513) and GAPDH (1:3,000 dilution; product code ab181602; both from Abcam) overnight at 4°C and then HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5,000 dilution; product code ab205718; Abcam) was added for 2 h at room temperature. Finally, the protein bands were observed using an enhanced chemiluminescence reagent (EMD Millipore) and analyzed by ImageJ software (v1.8.0; National Institutes of Health).
3
Immunohistochemical Analysis of HOXAs
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The sections were dewaxed, antigen-retrieved, and blocked. Then, specific antibodies (anti-HOXAs: HOXA1: ab230513; HOXA2: ab229960; HOXA3: ab230879; HOXA10: ab191470, Abcam) covered the sections at 4 °C overnight. After washing three times with PBS, specific secondary antibodies covered the sections for 1 h at 37 °C with horseradish peroxidase, then immersed in diaminobenzidine (DAB) and counterstained with hematoxylin for 2 min. A microscope (BX50/BX-FLA/DP70, OLYMPUS) was used to observe the staining signals. ImageJ Pro (Media Cybernetics, USA) was used by a technician (blinded to the experimental groupings) to quantify the integrated optical densities.
4
Protein Expression Analysis by Western Blot
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Pre‐lysed in radio‐immunoprecipitation assay cell lysis buffer (Gibco), proteins from cells and tissues were extracted by centrifugation and detected by bicinchoninic acid kit (Sangon Biotech Co., Ltd., Shanghai, China). Successively separated on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, the protein samples were blocked with 5% skim milk, reacted with primary antibodies HOXA1 (1:1000, ab230513, Abcam, Cambridge, UK), LC3‐II and LC3‐I (1:1000, ABC929, Sigma‐Aldrich, CA, USA) and GAPDH (1:2500, ab9485, Abcam). Next, the protein samples were incubated with secondary antibody (1:1000, ab150077, Abcam) and developed by enhanced chemiluminescence (Millipore, MA, USA) to detect gene protein expression.14
5
Immunohistochemical Analysis of HOXA1 Expression in Mouse Hippocampal Tissues
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The hippocampal tissues obtained from five mice in each group were fixed with 4% polyformaldehyde, paraffin embedded, and sliced into 4-μm serial sections. The sections were then deparaffinized, followed by antigen retrieval. The sections were stained using Histostain SP-9000 immunohistochemistry reagent (Zymed, San Francisco, CA, USA) and incubated overnight at 4°C with primary rabbit antibody against HOXA1 (ab230513; 1:500; Abcam, Cambridge, UK) after which they were subjected to incubation at 37°C for 30 min with secondary antibody (ab6728; 1:1,000; Abcam, Cambridge, UK). After PBS washing and incubation with HRP-labeled working solution, the sections were developed using diaminobenzidine, counterstained with hematoxylin, and mounted. The tissues that were stained in brown around the cytoplasm viewed using an optical microscope (Nikon, Tokyo, Japan) were considered to be positive. The sham-operated mice were regarded as the NC.
6
Immunohistochemical Analysis of HOXA1 in NPC
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Formalin-fixed, paraffin-embedded NPC tissue specimens were analyzed by immunohistochemistry as previously described (20 (link)). Briefly, a primary antibody against HOXA1 (ab230513; rabbit polyclonal; working dilution 1:100; Abcam, Cambridge, UK) was used for immunohistochemistry assays, and a non-biotin horseradish peroxidase detection system (DAKO, Glostrup, Denmark) was used to detect the expression level of HOXA1 protein. Both the extent and intensity of immunostaining were taken into consideration when analyzing the data. The intensity of staining was scored from 0 to 3, and the extent of staining was scored from 0% to 100%. The final quantitation of each stain was obtained by multiplying the two scores. HOXA1 expression was classified as high if the score was higher than 1.5, whereas scores of 1.5 or less indicated low expression.
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