Mammary glands were fixed overnight in 4% paraformaldehyde, washed with PBS and then processed and embedded in paraffin. Paraffin-embedded tissues were sectioned at 4-μm. Tissue sections deparaffinized in xylene, and then rinsed in ethanol and washed in water. Antigen retrieval was performed by heating the slides in 0.01 M citrate buffer (pH 6.0) in a microwave. The sections were cooled naturally to room temperature (RT) and then blocked for 1 hour at RT with blocking solution. The sections were incubated with primary antibodies overnight at 4 °C and incubated with secondary antibodies (Life Technologies) for 1 hour at RT. The slides were then washed 3 times with PBS and stained with DAPI for 5 min. The sections were covered with mounting medium. Antibodies against the following proteins were used: keratin 8 (Abcam, Ab59400, 1:400), keratin 14 (Abcam, Ab9220, 1:400), GFP (Abcam, Ab13970, 1:1000), keratin 5 (Covance, 905501, 1:800), Ki67 (Abcam, Ab16667, 1:1000), NFAT2 (Abcam, Ab2796, 1:400), and RFP (Invitrogen, MA5-15257).
Ab9220
Manufactured by Abcam
Sourced in United Kingdom
Ab9220 is a laboratory reagent. It is a monoclonal antibody that recognizes a specific target protein. The core function of Ab9220 is to bind and detect the target protein for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Ab59400, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Ma5 15257, by Thermo Fisher Scientific (1 mentions)
Ab13970, by Fortrea (1 mentions)
Ab80522, by Abcam (1 mentions)
Ab134179, by Abcam (1 mentions)
Ab52625, by Abcam (1 mentions)
Ab81289, by Abcam (1 mentions)
Enhanced chemiluminescence plus, by GE Healthcare (1 mentions)
Hrp conjugated anti mouse igg, by GE Healthcare (1 mentions)
Mouse anti gfp, by Beyotime (1 mentions)
Rabbit anti stat3, by Santa Cruz Biotechnology (1 mentions)
Mouse anti β actin, by Beyotime (1 mentions)
Protran nitrocellulose membrane, by Merck Group (1 mentions)
Rabbit anti foxm1, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg, by GE Healthcare (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
5 protocols using ab9220
1
Mammary Gland Tissue Immunohistochemistry
2
Isolation and Characterization of Human Epidermal Stem Cells
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Human foreskin samples were obtained from seven healthy donors. Primary EpSCs were isolated using a standard protocol adopted from Yang et al. [15 (link)], which allowed the retention of many characteristics of EpSCs, including morphology and antigens. Flow cytometry and immunofluorescence (IF) staining with antibodies of K19 (1 : 200 diluted, ab52625, Abcam, UK), K15 (1 : 100 diluted, ab80522, Abcam), K14 (1 : 200 diluted, ab9220, Abcam), CD34 (1 : 200 diluted, ab81289, Abcam), and β1-integrin (1 : 250 diluted, ab134179, Abcam) were, respectively, performed to identify the phenotype of our EpSCs. Finally, isolated cells were cultured in a Keratinocyte medium (KM, 2101, cell science) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, in a humidified atmosphere of 95% air and 5% CO2 at 37°C. All the participants wrote the informed consent, and the experimental procedures were approved by the Ethical Review Board at the First Affiliated Hospital of Zhejiang Chinese Medicine University (No. 2017-KL-024-01).
3
Protein Expression Analysis by Western Blot
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To measure protein levels, cell lysates were resolved by denaturing gel electrophoresis before electrotransfering to Protran nitrocellulose membrane (Millipore, USA). The membrane was subjected to Western blot analysis with antibodies against proteins of interest. The signals from the primary antibody were amplified by horse radish peroxidase (HRP)-conjugated anti-mouse IgG (GE, USA) or anti-rabbit IgG (GE, USA), and detected with Enhanced Chemiluminescence Plus (GE, USA). The following antibodies and dilutions were used: rabbit anti-Foxm1 (1∶500; Santa cruz SC-502), rabbit anti-Nanog (1∶1000; Millipore AB9220), rabbit anti-Sox2 (1∶1000; Abcam AB59776), mouse anti-Oct4 (1∶1000; Cell signaling #4286), rabbit anti-Stat3 (1∶500; Santa cruz SC-482), rabbit anti-p-Stat3 (1∶500; Santa cruz SC-8059), rabbit anti-Klf4 (1∶500; Santa cruz SC-20691), mouse anti-β-actin (1∶5000; Beyotime AA128), mouse anti-GFP (1∶1000; Beyotime AG279).
4
Immunohistochemical Analysis of Lung Tissue
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Lung regions were fixed in 4% paraformaldehyde, placed in sequential sucrose solutions, and embedded in OCT for freezing. Tissues were sectioned using a microtome. GFP positive images were quantified using ImageJ. The percentage of manually counted GFP positive cells was calculated as a ratio to the number of DAPI positive cells. Frozen sections were probed with anti-acetyl tubulin at 1:200 dilution (Cell Signaling Technology, D20G3, K40, Danvers, MA) and anti-human cytokeratin 5, 8 (CK5, 8) polyclonal antibody at 1:5 dilution (US Biological Life Sciences, 168994, Salem, MA). Basal cells were detected by anti-nerve growth factor receptor (NGFR) (1:100 dilution) (ThermoFisher Scientific, 14-9400-80, Waltham, MA, USA) and anti-cytokeratin 14 (CK14) (1:100) (Abcam, ab9220, Cambridge, UK). CFTR was detected by immunohistochemistry and immunofluorescence using mouse monoclonal CFTR 769 from the UNC/CFF consortium (29–31 (link)). Slides were counterstained and mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA).
5
Localization of WNT5A in Keloid Tissues
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Double-immunofluorescence staining of WNT5A (1:200; ab229220; Abcam) with cytokeratin 14 (1:200; ab9220; Abcam), CD31 (1:800; #3528; Cell Signaling Technology) and α-SMA (1:100; 14–9760-82; Invitrogen) was performed to determine cell localization and differential expression of WNT5A in keloid and normal tissues. Sections were observed using fluorescence microscopy (confocal LSM 700; Zeiss, Oberkochen, Germany).
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