Formalin-fixed HCC tissue samples were used to perform this assay according to the manufacturer’s instruction. After incubation with anti-CELSR2 primary antibody (Cell Signaling Technology, USA) and anti-glyceraldehyde phosphate dehydrogenase (GAPDH) primary antibody (Abcam, USA), the sections were then incubated with the corresponding secondary antibody (Abcam) for 1 h at room temperature. Phosphate buffered saline (PBS) was used as negative controls (NC). Semiquantitative analysis of the obtained images was performed using the Image-Pro Plus 6.0 software. A score, calculated by multiplying the staining intensity by the area of positively-stained cells, was assigned for each image.
Corresponding secondary antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Corresponding secondary antibody is a laboratory reagent used to detect and amplify the signal from a primary antibody in immunoassays and other applications. It binds specifically to the Fc region of the primary antibody, allowing for visualization or quantification of the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Gapdh primary antibody, by Abcam (1 mentions)
Radio immunoprecipitation assay ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Nbt bcip reagent kit, by Thermo Fisher Scientific (1 mentions)
Anti jak1, by Abcam (1 mentions)
Anti caveolin 1, by Abcam (1 mentions)
Quantity one system, by Bio-Rad (1 mentions)
Anti caspase 3, by Abcam (1 mentions)
Anti p stat5, by Abcam (1 mentions)
Anti pp2a, by Abcam (1 mentions)
Anti pias1, by Abcam (1 mentions)
Anti pias3, by Abcam (1 mentions)
Anti stat5, by Abcam (1 mentions)
Anti socs1, by Abcam (1 mentions)
Anti jak3, by Abcam (1 mentions)
Anti pim1, by Abcam (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
11 protocols using corresponding secondary antibody
1
Immunohistochemical Analysis of CELSR2 in HCC
2
Protein Extraction and Western Blot
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Proteins were extracted from the tissue or cells of five mice using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Thermo Fisher Scientific, USA) to collect total protein using five mice separately which was separated on a 12.5% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Sigma-Aldrich, USA) in turn. The PVDF membrane was blocked by 1% BSA. Then it was incubated with specific primary antibody made by Bioss Antibodies company (Bioss, China) and then with corresponding secondary antibody (Abcam, UK). Finally, the PVDF membrane was stained using NBT/BCIP Reagent Kit (Thermo Fisher Scientific, USA).
3
Quantification of Colon Tissue Proteins
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Colon tissues (n = 6) were quickly shredded and mixed with protease inhibitor cocktails, which were randomly selected from every group. The mixture was homogenized by ultrasound on crushed ice and centrifuged at 13000g at 4°C for 10 min; the supernatant was extracted; and the protein concentration was determined by BCA protein assay. Equal amounts of total protein (60 μg) were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane using a semidry transfer slot (Bio-Rad, USA). After blocking with 5% nonfat dried milk for 1 h, the membrane was incubated with proportionally diluted antibodies at 4°C overnight. The antibodies included rabbit anti-GAPDH (1:2,000), anti-BCL-2 (1:2,000), anti-BIM-1 (1:1,000), anti-caspase-3 (1:500), anti-BAX (1:1,000), anti-PP2A (1:1,000), anti-β-casein (1:2,500), anti-caveolin-1 (1:1,000), anti-Pim-1 (1:1,000), anti-JAK1 (1:1,000), anti-JAK3 (1:1,000), anti-PIAS1 (1:2,000), anti-PIAS3 (1:2,000), anti-Socs-1 (1:1,000), anti-P-STAT5 (1:1,000), and anti-STAT5 (1:1,000; Abcam). Then, samples were incubated with a corresponding secondary antibody (Abcam) for 1 h. After washing three times with TBST, the blots were visualized by the Proteogel imaging system (FluorChem M, ProteinSimple, USA) and quantified with the Quantity One System (Bio-Rad).
4
Western Blot Protein Analysis Protocol
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The assay was implemented in line with the previous description [23 (link)]. Generally, the separated proteins were and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, U.S.A.). Then, the membranes were blocked with 5% non-fat milk. Afterwards, the membranes were incubated with primary antibodies, including ZEB1 (1:1000, ab181451; Abcam, Cambridge, MA, U.S.A.), P-gp (1:1000, ab3364; Abcam) and GST-π (1:800, ab47709; Abcam) and GAPDH (1:8000, ab8245, Abcam), followed by probed with corresponding secondary antibody (Abcam) to combine these primary antibodies. At last, proteins blots were assessed with an enhanced chemiluminescence detection kit (Millipore).
5
Quantification of Myocardial Protein Expression
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The total protein in the myocardial tissue of mice was extracted and quantified by bicinchoninic acid method (Boster, Hubei, China). After sodium dodecyl sulphate polyacrylamide gel electrophoresis, the protein was transferred to a polyvinylidene fluoride membrane and sealed with 5% bovine serum albumin. The membrane was cultured with primary antibodies Myd88 (1:1000), Bcl-2 (1:2000), Bax (1:2000) and GAPDH (1:3000) (all from Abcam, MA, USA) and with corresponding secondary antibody (MTBio, Shanghai, China). The plots were developed by chemiluminescence reagent, observed by Bio-rad Gel Doc EZ imager (Bio-rad, CA, USA) and analyzed by ImageJ software (Gao et al. 2015 (link)).
6
Immunofluorescence Staining of Spinal Cord
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After fixation, the spinal cords were embedded and frozen in CRYO-OCT compound (Tissue-Tek, Torrance, California) and then sectioned into slices of 5 µm in thickness. The slices were fixed with ice-cold acetone for 10 minutes and dried in the air for 30 minutes. Then, the slices were rinsed with PBS for 2 minutes and blocked with 2% bovine serum albumin (BSA) in PBS for 30 minutes at room temperature. The slices were then incubated overnight at 4°C with mouse anti-rat CD31 (1:200, Abcam, San Francisco, California) to trace the resting endothelial cells or CD61 antibody (1:200, Abcam) to trace the activated neovascular endothelial cells. Then, the slices were incubated with Cy3 and immunoglobulin G-conjugated secondary antibody (1:600, Abcam) for labeling. After 3 more washes with PBS, the slides were stained for nuclei with 4′,6-diamidino-2-phenylindole (DAPI). The immunofluorescence staining was observed under an epifluorescence microscope. Staining against βIII tubulin was performed with anti-βIII tubulin antibody (1:200, Abcam) and corresponding secondary antibody (1:600, Abcam). Ten random visual field areas within 500 µm around the damage center were selected for quantification. The fluorescence was quantified with the software of image-Pro Plus 6.0.
7
Protein Extraction and Western Blot Analysis
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Heart tissue samples (50-100 mg) and cardiomyocytes samples were collected and were fully lysed with RIPA lysate (Cell Signaling Technology, USA) for 30 min, and the protein concentration was detected with BCA protein detection kit. After 10% SDS-PAGE gel electrophoresis, the protein was transferred to PVDF membrane, and 5% skimmed milk powder blocked the protein at room temperature for 2 hours. The specific primary antibody (Abcam, UK) was incubated with PVDF membrane overnight at 4°C, and the corresponding secondary antibody (Abcam, UK) was incubated with PVDF membrane at room temperature. Chemiluminescence was developed using a hypersensitive ECL kit (Baiaosi, China).
8
ATAD2 Protein Detection in Colon Cancer Cell Lines
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Firstly, the proteins were obtained from SW480 and SW620 cells lysed by RIPA lysis buffer (Beyotime, Shanghai, China). Next, the proteins were subjected to 10% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). After blocked by 5% TBST containing 5% non-fat skim for 2 h, the membranes were incubated with the primary antibody against ATAD2 or GAPDH (1:1000; Abcam, Cambridge, MA, USA) at 4°C overnight, and then incubated by corresponding secondary antibody (1:400; Abcam) for 1 h. Finally, the ECL Western blot kit (Beyotime) was used for the measurement of protein bands.
9
Immunofluorescence Protocol for Brain Sectioning
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Immunofluorescence was carried out as previously reported (Zhang et al., 2020 (link)). Coronal brain sections (30 μm) were made using a cryotome (Leica). The brain slices were washed with PBS and permeabilized using 0.3% Triton X-100. After that, 10% goat serum was used for slice blocking. Primary antibody solutions were made according to the dilution rate provided by manufacturers and applied to the slice incubations at 4°C overnight with gentle shaking. The primary antibodies and dilutions used in the article were as follows: GSDMD (Abcam, ab219800), 1:100; Iba-1 (Sigma, SAB2702364), 1:100; GFAP (CST, 3670), 1:200; NeuN (Millipore, MAB377), 1:100; CD68 (Abcam, ab125212), 1:500; GFAP (Abcam, ab7260), 1:300; MAP2 (Abcam, ab32454), 1:500. The next day, the slices were rewarmed at 37°C for 1 h and washed three times with PBS. Corresponding secondary antibodies (Abcam) were diluted and used for staining following by nuclear labeling using DAPI (10 μg/ml, Sigma). Finally, the slices were washed and mounted using antifade mounting medium (Santa). Then, the slices were imaged with confocal microscopy (Leica) and analyzed using ImageJ software (NIH).
10
Western Blotting Analysis of UPR Markers
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Cells were collected and suspended in a RIPA lysis buffer system (Santa Cruz) and whole-cell extract was prepared. Total protein was extracted using the Protein Extraction kit (Beyotime) according to the instructions provided by the manufacturer. A BCA kit (Beyotime) was used to measure the protein concentrations. The protein was separated by vertical SDS-PAGE and then transferred to the PVDF membranes electronically. The blocking buffer (Santa Cruz) was applied to the membranes. Primary antibodies against GRP78 (1: 2000; Abcam), PERK (1: 2000; CST), p-PERK (1: 2000; CST), cleaved ATF6 (1: 2000; Abcam), IRE-1α (1: 4000; CST), p-IRE-1α (1: 2000; CST), CHOP (1: 2000; Abcam), caspase12 (1: 2000; Abcam), and GAPDH (1: 4000; Sigma) were used to incubate the membranes at 4°C for 8 h in TBST. Then, the corresponding secondary antibodies (Abcam) were used to incubate the membranes at room temperature for 1 h. The membranes were developed by an ECL kit (pierce) and then exposed with the Gene Genius system (Syngene). The densities of the blots were then analyzed with Image J software.
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