6 mm excisional wounds were generated as previously described (73 (link)). After 10 days, wounds were prepared for histological evaluation using the following procedure. Excised wounds were fixed by placing them in 4% paraformaldehyde for 24 hours, following fixation the wound was placed in a cassette that allowed for the dehydration of the tissue, followed by clearing of the tissue using xylene (Fisher brand), and finally imbedding the tissue in paraffin wax. Sections (5 μm) of the paraffin block were placed on clear glass slides for further treatment and staining. Staining and probing for Masson’s trichrome, Picrosirius red, Hsp47 (rabbit polyclonal anti-Hsp47 (Abcam ab109117); 1:4000), rabbit polyclonal anti-Fibroblast activating protein (Abcam ab53066); 1:200), and mouse polyclonal anti-type I collagen (Millipore AB765P); 1:1000) were performed by Histo-Scientific Research Laboratories Inc.
Ab109117
Manufactured by Abcam
Sourced in United Kingdom
Ab109117 is a monoclonal antibody that recognizes the Cytokeratin 14 protein. Cytokeratin 14 is a type I intermediate filament protein expressed in basal cells of stratified squamous epithelia.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab53066, by Abcam (1 mentions)
Xylene, by Thermo Fisher Scientific (1 mentions)
Ab765p, by Merck Group (1 mentions)
Ab16821, by Abcam (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ab18203, by Abcam (1 mentions)
As 1111, by Aspen (1 mentions)
As1018, by Aspen (1 mentions)
Wet transfer system, by Bio-Rad (1 mentions)
Ecl reagent, by Wuhan Servicebio Technology (1 mentions)
Ripa lysis buffer, by Wuhan Servicebio Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab37150, by Abcam (1 mentions)
Ab49254, by Abcam (1 mentions)
9 protocols using ab109117
1
Histological Evaluation of Excisional Wound Healing
2
Western Blot Analysis of Tumor Proteins
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T6-17 cells were grown to 80% confluence on 10 cm plates. The plates were washed twice with PBS and then incubated on ice for five minutes in 1 mL RIPA Buffer (Sigma-Aldrich) supplemented with 6 M urea. Cells were scraped off the plate and clarified by centrifugation. 47 mg of solid tumor was solubilized in 3 mL Western Lysis Buffer (12.5 mM Tris, 4% SDS, pH 8) with a mortar and pestle. Lysate was boiled for 30 min and clarified by centrifugation. Total protein concentrations were determined by BCA Assay (Pierce). Hsp47, integrin, and ErbB2 were analyzed by Western blot. Blots were incubated for 1 hr with Odyssey Blocking Buffer (Li-Cor), washed 3 times with TBS-T, stained with antibodies against Hsp47, Integrin αV, and ErbB2 (AbCam ab109117, ab16821, and ab8054) overnight at 4C, washed 3 times with TBS-T, stained with fluorescently-labeled anti-rabbit and anti-mouse secondary antibodies (Li-Cor) for 1 hr at room temperature, washed 3 times with TBS-T, and imaged on a Li-Cor Odyssey.
3
Immunofluorescence Staining of Cell Markers
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We fixed the cells by incubating in 4% PFA solution (AS1018, Aspen, Wuhan) for 30 min. Then, after blocking the fixed cells with 10% BSA (10735078001, Roche) at 37 °C for 30 min, they were incubated at 4 °C overnight with the following primary antibodies: anti-SERPINH1 (ab109117, Abcam); anti-E-cadherin (20874-1-AP, San Ying, Wuhan); and anti-N-cadherin (Abcam, Ab18203). Then, the cells were incubated with CY3- or FITC-conjugated goat anti–mouse or goat anti–rabbit IgG (AS-1111, Aspen, Wuhan) at 37 °C for 1 h. The cells were imaged and analyzed under a fluorescence microscope.
4
Western Blot Protein Analysis Protocol
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Cells were lysed by RIPA lysis buffer (Servicebio, Wuhan, China) after washing with ice-cold PBS. Protein concentrations were determined using BCA Protein Assay Kit (Thermo Fisher Scientific Inc, USA). The extracted proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA) using a wet transfer system (Bio-Rad Laboratories, USA). Membranes were blocked in TBST buffer containing 5% nonfat milk for at least 1 h and incubated with primary antibodies (anti-GAPDH, Abcam, ab9485; anti-SERPINH1, ab109117) overnight at 4 °C. The next day, following incubation with an appropriate secondary antibody, immunoreactions on the PVDF membrane were detected with an ECL reagents (Servicebio, Wuhan, China).
5
Protein Expression Analysis in Gastric Cancer
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We extracted total proteins from human GC tissues and cell lines using the RIPA lysis buffer (AS1004, ASPEN, Wuhan). Then, equal amounts of the protein lysates were separated on a 10% SDS PAGE. The resolved proteins were transferred onto PVDF membranes (IPVH00010, Millipore) and blocked with 5% non-fat milk in TBST for 2 h at room temperature. Then, the membrane was incubated overnight at 4°C with the following primary antibodies: anti- SERPINH1 (ab109117, Abcam), anti-Wnt2 (sc-514382, SANTA CRUZ), anti-β-Catenin (17565-1-AP, SanYing, Wuhan), anti-p-GSK3β (#5558, CST), anti-GSK3β (#12456, CST), anti-NFκB p65 (#8242, CST), anti-Snail1 (13099-1-AP, SanYing, Wuhan), anti-Slug (ab106077, Abcam), anti-TWIST (ab49254, Abcam), anti-MMP2(ab37150, Abcam), anti-MMP9 (ab76003, Abcam), anti-E-Cadherin (#3195, CST), anti-N-Cadherin (#4061, CST). Subsequently, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (AS1107, ASPEN). The blots were developed using the enhanced chemiluminescence system (AS1059, ASPEN) and the protein bands were imaged.
6
Immunohistochemical Staining of Tissue Samples
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Immunohistochemistry (IHC) staining was conducted as previously reported [22 (link)]. In brief, tissue slices were first deparaffinized and gradually rehydrated using a series of graded ethanol solutions. To facilitate antigen retrieval, the deparaffinized tissue slices were subjected to boiling in 10 mM citrate buffer (pH 6.0) for 20 minutes. Afterwards, the tissue slices were incubated with 3% H2O2 solution to quench the activity of endogenous peroxidase. The slices were immersed with the specific primary antibody (1:500 dilution, ab109117, Abcam). Visualization of the antibody-antigen interaction was achieved through DAB, followed by counterstaining with hematoxylin.
7
Immunocytochemistry of Cardiac Fibroblasts
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AMCFs were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 for 20 min, and then blocked with 5% BSA in PBS-Tween for 1 h. Subsequently, AMCFs were diluted with α-SMA antibody (Abcam, ab5694) or HSP47 antibody (Abcam, ab109117) in 5% BSA at 4 °C overnight. DAPI was used to counterstain the nuclei.
8
Western Blot Immunodetection Procedure
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Proteins for immunoblotting were resolved by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% skimmed milk and then incubated with SERPINH1 rabbit polyclonal antibody (ab109117, Abcam, Cambridge, UK; 1:2,000 dilution; RRID: AB_10888995) and GAPDH rabbit polyclonal antibody (#2118, Cell Signaling Technology, Boston, MA, USA; 1:5,000; RRID: AB_561053) at room temperature. The membrane was incubated with horseradish peroxidase-conjugated sheep anti-rabbit IgG secondary antibody (ab6721, Abcam; 1:2,000 dilution; RRID: AB_955447) at room temperature for 2 h, and then washed 3 times with phosphate-buffered saline with Tween 20 (PBST, PBS contained 0.1% Tween 20) for 20 min. Chemiluminescent signal was visualized using a ClarityTM Western ECL Substrate (#170-5061, Bio-Rad Laboratories, Inc.,) and detected by Tanon 5200 full automatic chemiluminescence image analysis system (Tanon Science and Technology Co., Ltd., Shanghai, China).
9
Western Blot Immunodetection Protocol
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Proteins for immunoblotting were resolved by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% skimmed milk and then incubated with SERPINH1 rabbit polyclonal antibody (ab109117, Abcam, Cambridge, UK; 1:2000 dilution) and GAPDH rabbit polyclonal antibody ( # 2118,Cell Signaling Technology, Boston, USA; 1: 5000) at room temperature. The membrane was incubated with horseradish peroxidase-conjugated sheep anti-rabbit IgG secondary antibody (ab6721, Abcam, Cambridge, UK; 1:2000 dilution) at room temperature for 2 hours, and then washed 3 times with PBST buffer (PBS contains 0.1% Tween 20) for 20 minutes. Chemiluminescent signal was visualized using a ClarityTM Western ECL Substrate ( # 170-5061, Bio-Rad Laboratories, Inc., Hercules, CA, USA) and detected by Tanon 5200 full automatic chemiluminescence image analysis system (Tanon Science and Technology Co., Ltd., Shanghai, China).
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