Primary antibodies against Snail1 (cat. no. 125918) were purchased from GeneTex, Inc. (Irvine, CA, USA). Primary antibodies against Wnt1 (ab85060), Wnt8a (ab130930), phosphorylated glycogen synthase kinase 3β (P-GSK3β; ab130937) and GAPDH (ab37168) were obtained from Abcam (Cambridge, UK). Primary antibodies against Wnt3a (bs1700R) and Wnt5a (bs1948R) were purchased from BIOSS (Beijing, China). The primary antibody against Wnt11 (sc50360) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies against β-catenin (BA0426) and β-actin (BM0627) were obtained from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). The antibody against GSK3β (22104-1-AP) was purchased from Wuhan Sanying Biotechnology (Wuhan, China). The bicinchoninic acid (BCA) protein assay kit was purchased from Beyotime Institute of Biotechnology (Haimen, China). TRIzol (252250AX) was obtained from Aidlab Biotechnologies Co., Ltd. (Beijing, China). M-MLV Reverse Transcriptase (RNase H; CO2010A) and ddH2O (DNase/RNase Free; C1D230A) were purchased from GeneCopoeia, Inc. (Rockville, MD, USA) and RNase Inhibitor (I21222) was obtained from TransGen Biotech, Inc. (Beijing, China).
Ab85060
Manufactured by Abcam
Sourced in United Kingdom
Ab85060 is a recombinant protein produced in E. coli. It has a molecular weight of approximately 25 kDa and is supplied as a lyophilized powder.
Automatically generated - may contain errors
Lab products found in correlation
Ab16663, by Abcam (2 mentions)
Ab220962, by Abcam (2 mentions)
Sc50360, by Santa Cruz Biotechnology (1 mentions)
Ab37168, by Abcam (1 mentions)
Bs 1700r, by Bioss Antibodies (1 mentions)
Bs1948r, by Bioss Antibodies (1 mentions)
Bm0627, by Wuhan Boster Biological Technology (1 mentions)
Bicinchoninic acid bca protein assay kit, by Beyotime (1 mentions)
Ab55906, by Abcam (1 mentions)
Enhanced chemiluminescence method, by Beyotime (1 mentions)
Ab196204, by Abcam (1 mentions)
Peroxide conjugated secondary antibody, by Beyotime (1 mentions)
Ab64693, by Abcam (1 mentions)
Bx50 bx fla dp70, by Olympus (1 mentions)
Ab32572, by Abcam (1 mentions)
C1002, by Beyotime (1 mentions)
Gapdh, by BD (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Keygen Biotech (1 mentions)
Pvdf membrane, by Keygen Biotech (1 mentions)
5 protocols using ab85060
1
Western Blot Analysis of Wnt Pathway Proteins
2
Western Blot Analysis of Wnt Pathway
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Cells were harvested, washed twice by ice-cold PBS, and subjected to lysis. Proteins (20 µg) from cell lysates were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk and immunoblotted with primary antibodies (rabbit anti-mouse Axin1 (ab55906, Abcam), rabbit anti-mouse Wnt1 (ab85060, Abcam), rabbit anti-mouse β-catenin (ab196204, Abcam)) overnight at 4°C followed by incubation at room temperature with a peroxide-conjugated secondary antibody (Beyotime, China). Proteins were then visualized by an enhanced chemiluminescence method (Beyotime, China) according to the manufacturer’s instructions.
3
Wnt1 Pathway Modulation in SAH Neuroprotection
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Brain tissue samples were obtained at corresponding time points after SAH. Interventions of rhwnt1, siwnt1 RNA, and anti-Frizzled1 were applied once SAH models were established. Expression of Wnt1, Frizzled1, β-catenin, and CD36 in neurons was measured by immunofluorescent double staining. CD206-positive cells were identified as M2-type microglia. First, brain samples were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm sections. Primary antibodies (1:100) of Wnt1 (rabbit polyclonal antibody, ab85060; Abcam), Frizzled1 (goat polyclonal antibody, sc-30428; Santa Cruz Biotechnology Inc.), β-catenin (rabbit monoclonal antibody, ab32572; Abcam), CD206 (rabbit polyclonal antibody, ab64693; Abcam), and CD36 (rabbit polyclonal antibody, ab64014; Abcam) were added and incubated for 12 hours at 4°C. Sections were washed three times, and then the corresponding secondary antibodies (1:500 dilution) were added and incubated for 1 hour. Finally, sections were covered with anti-fading mounting medium containing 4′,6-diamino-2-phenyl indole (DAPI) (C1002; Beyotime) and observed using a fluorescence microscope (BX50/BX-FLA/DP70; Olympus, Tokyo, Japan). Fluorescence intensity attached to different groups was analyzed using ImageJ program 1.8.0 version and normalized to fluorescence intensity of the sham group.
4
Protein Extraction and Western Blot Analysis
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The proteins were extracted by using Radio-Immunoprecipitation Assay (RIPA) (Beyotime Institute of Biotechnology, CHN) lysis buffer adding with PMSF (KeyGEN, CHN) and protease inhibitor cocktail. Forty micrograms of protein lysates were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel (KeyGEN, CHN) and transferred to a polyvinylidene fluoride (PVDF) membrane (KeyGEN, CHN). After blocking with skimmed milk for 1 hour, the membrane was incubated with the specific primary antibodies overnight. The membranes were then washed and incubated with secondary antibodies (ZSGB-BIO, CHN) at room temperature for 2 hours. Antibodies against Wnt1 (1500, ab85060), cyclin D1 (1100, ab16663), SPINDOC (1:50, ab220962) were purchased from Abcam (UK). β-catenin (1:500, 610153), GAPDH (1:5000, 60004-1-Ig) were purchased from BD sciences (USA). AXIN2 (1:50, K006954P) and SPIN1 (1:50, K006346P) were purchased from Solarbio (CHN).
5
Immunohistochemical Analysis of Tumor Markers
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Fresh nude mouse tumor specimens were converted into wax blocks by routine fixation, and standardized operation was carried out according to the kit instructions (Beijing Dingguo Changsheng Biotechnology, CHN). The primary antibody was added and incubated at 4°C overnight. The specific information of primary antibodies used is as follows: Wnt1, 1:500, ab85060, Abcam, (UK); cyclin D1, 1:100, ab16663, Abcam, (UK); SPINDOC, 1:50, ab220962, Abcam, (UK); β-catenin,1:500, 610153, BD sciences, (USA); AXIN2, 1:50, K006954P, Solarbio, (CHN); SPIN1, 1:50, K006346P, Solarbio, (CHN). The blocks were washed with PBS 3 times, 2min each, and biotin-labeled sheep anti-rabbit IgG solution (1:100, Z021006Y, ZSGB-BIO, CHN), was added followed by incubation at room temperature for 30min. Thereafter, DAB chromogenic solution was added after PBS washing, and the blocks were placed under the microscope for observation. The reaction time was controlled, and the blocks were then washed with the distilled water to stop the reaction. Hematoxylin (Beijing Dingguo Changsheng Biotechnology, CHN) was added for redyeing, followed by washing, routine dehydration, spin drying, and observation of the results under the microscope after sealing with the neutral adhesive.
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