Standard Western blotting was conducted for protein expression assays from MG-63 cells with miR-373 mimic, inhibitor, and miR-control. Briefly, proteins were isolated with RIPA lysis buffer containing 1 mg of protease inhibitors (Applygen Technologies Inc., Beijing, P.R. China) after 2 days of transfection. The protein content was quantified using Bicinchoninic Acid (BCA) Protein Assay Kit (CoWin Biotech Co., Ltd., Beijing, P.R. China). The following primary antibodies p53 (ab1101), p21 (ab109520), p53 upregulated modulator of apoptosis (Puma; ab9643), B-cell lymphoma-2 associated X (Bax; ab32503), plasminogen activator inhibitor (PAI; ab66705), PI3K (ab86714), AKT (ab8805), Rac1; ab33186), c-Jun N-terminal kinase (JNK; ab124956), and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Abcam (Cambridge, UK). Subsequently, secondary antibodies were marked by horseradish peroxidase for 2 h at 37°C. Samples were then electrotransferred to polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Boston, MA, USA). The bands were visualized by the Odyssey CLx equipment (LI-COR Bioscences, Lincoln, NE, USA).
Ab66705
Manufactured by Abcam
Sourced in United Kingdom
Ab66705 is an antibody product from Abcam. It is a primary antibody that targets a specific protein or antigen. This product is intended for use in various laboratory applications, but no further details about its intended use or specific applications are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab2413, by Abcam (3 mentions)
α sma, by Thermo Fisher Scientific (3 mentions)
Ab103791, by Abcam (2 mentions)
Plasminogen, by Merck Group (2 mentions)
Ab14715, by Abcam (2 mentions)
Ab1791, by Abcam (2 mentions)
D val leu lys 7 amido 4 methylcoumarin, by Merck Group (2 mentions)
Ab6992, by Abcam (2 mentions)
Irdye 800cw, by LI COR (2 mentions)
Ab34710, by Abcam (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ab124956, by Abcam (1 mentions)
Ab1101, by Abcam (1 mentions)
Ab33186, by Abcam (1 mentions)
Ab9643, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab109520, by Abcam (1 mentions)
Pvdf microporous membrane, by Merck Group (1 mentions)
Ab32503, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
23 protocols using ab66705
1
Investigating miR-373 Regulation in MG-63 Cells
2
Immunofluorescence Analysis of TLR2, Serpin E1, PLAUR, BNP, OSM, PGP9.5, NeuN, and PLAUR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin sections were de‐paraffinized, rehydrated, permeabilized, followed by incubation with rabbit antibody to TLR2 (1:300, Abcam ab213676), Serpin E1 (1:300, Abcam ab66705), PLAUR (1:300, Abcam ab103791), BNP (1:100, Abcam ab236101), OSM (1:75, Thermo PA576861), mouse antibody to PGP9.5 (1:300, Abcam ab8189), NeuN (1:500, Novus NBP1‐92693), TLR2 (1:300, Abcam ab16894), or Antigen Affinity‐purified polyclonal goat IgG against PLAUR (10ug/ml, AF534, R&D) in blocking solution (4°C, overnight). The samples were washed in PBS and incubated with donkey anti‐rabbit Alexa 594 (1:500, Abcam ab150064) or anti‐mouse Alexa 488 (1:500, Abcam ab150109). Subsequent to a final wash, specimens were mounted onto slides using prolonged anti‐fade reagents containing DAPI (ThermoFisher Scientific) and captured using an IX73 Olympus microscope. Fluorescence intensity was analyzed using CellSens Dimension Imaging software and Image J.
3
Inflammatory Marker Expression in Cardiac and Pulmonary Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHC staining was performed on samples from patients 2, 3, and 7 (Table 1 ). Sections from lungs and heart were analyzed for presence of macrophages (CD68), neutrophils (MPO), T cells (CD3), and B cells (CD20). The inflammatory markers, plasminogen activator inhibitor-1 (PAI-1), presently also denoted serpine-1, and monocyte chemoattractant protein (MCP-1/CCL2), were also examined. The tissue samples were deparaffinized, rehydrated and demasked in a microwave oven for 24 min in Target Retrieval Solution or Tris/Edta buffer pH 9.1 for ab CD 68 and CD3. The primary antibodies used were anti CD3 1:50 (rabbit polyclonal ab 5690, Abcam), anti CD20 1:2500 (confirm anti-CD20 (L26) monoclonal mouse anti CD ready-to-use (RTU) cat. no 760-2531 Roche Diagnostics), anti CD68 1:3000 (monoclonal mouse anti human CD68 cat. no M0814 Agilent Technologies), anti MPO 1:1000 (rabbit polyclonal cat. no A0398, Agilent Technologies), anti PAI-1 1:400 (rabbit polyclonal ab 66705, Abcam) and anti MCP-1 1:400 (rabbit polyclonal ab9669, Abcam). Antigen-antibody reactions were visualized with DAKO EnVision horse radish peroxidase system (Agilent Technologies or DAKO Cytomation for MCP-1) using 3, 3′-diaminobenzidin as the chromogen. All tissue sections were counterstained with hematoxylin.
4
Immunohistochemical Analysis of Tumor Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice tumour tissues were collected and made into 5‐μm paraffin‐embedded sections. Sections were dewaxed at 67°C, and then endogenous peroxidase was inactivated with 3% hydrogen peroxide methanol solution. Non‐specific antigens were blocked with 10% goat serum at 37°C for 1 hour. After washing thrice with PBS, sections were incubated at 37°C for 1.5 hours with primary antibodies, and then incubated with secondary antibody for 1 hour at room temperature. Then sections were washed with PBS and incubated with 3, 3′‐Diaminobenzidine (DAB; Sigma‐Aldrich) for 5 minutes in a dark room. After being washed with PBS, sections were then counterstained for 1 minute with hematoxylin (Sigma‐Aldrich). Permount™ Mounting Medium (Abcam) was utilized to mount the sections. Primary antibodies used were as listed below: rabbit anti‐SERPINE1 (5 μg/mL, ab66705; Abcam), rabbit anti‐p53 (20 μg/mL, ab1431; Abcam), rabbit anti‐MDM2 (1:100, ab131355; Abcam), rabbit anti‐cleaved Caspase3 (10 μg/mL, ab2302; Abcam) and rabbit anti‐ki67 (1:50, ab8191; Abcam). Secondary antibody was HRP‐labelled goat anti‐rabbit IgG (1:2000, ab205718; Abcam).
5
Immunofluorescence Analysis of PAI-1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen sections (6 μm thick) were cut from a cryostat and transferred onto gelatin-coated glass slides. The sections were fixed in sucrose-cacodylate buffer (0.1 mol/L sodium cacodylate, 0.1 mol/L sucrose, and 0.25% glutaraldehyde) for 15 minutes and permeabilised with 0.5% Triton X-100 in PBS. The sections were blocked with 1% bovine serum albumin in PBS at room temperature for 30 minutes. To detect the expression of plasminogen activator inhibitor-1 (PAI-1), the sections were incubated with primary antibodies against PAI-1 (ab66705) (1 : 500, Abcam, Cambridge, UK) followed by goat polyclonal antirabbit FITC-labelled (ab6717) secondary IgG antibodies (1 : 1000, Abcam). In partial staining, 4′,6-diamidino-2-phenylindole (DAPI) was used as a histological background control. Immunofluorescence imaging was performed manually with a 40x objective lens (camera: DP70; ISO: 200; Tv: 10 seconds for PAI-1) of an Olympus IX71 fluorescence microscope (Olympus, Tokyo, Japan).
6
Investigating PAI-1 and Fibronectin Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Verteporfin, human transforming growth factor (TGF-β1; T7039), plasminogen and D-Val-Leu-Lys-7-amido-4-methylcoumarin were from SigmaAldrich. OXA-06, PD-184352, SR-11302 were from Tocris. For protein detection specific antibodies against PAI-1 (Abcam Cat# ab66705, RRID:AB_1310540), fibronectin (abcam, ab2413), p-ERK-1/2 (Santa Cruz, E4, sc-7383), SDHA (abcam, ab14715) and histone H3 (abcam, ab1791) were used.
7
Immunohistochemical Analysis of Tongue OSCC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We obtained surgical specimens from 30 patients with primary tongue OSCC who were treated in the Department of Oral and Maxillofacial Surgery at Kyushu University Hospital from 2005 to 2018. The sections were deparaffinized in xylene and then hydrated by graded series of ethanol. The sections were then incubated with the following primary antibodies at room temperature: mouse anti-CD163 (Clone 10D6, 1:400 dilution; Novocastra, Newcastle, UK), rabbit anti-CD204 (ab217843, 1:200 dilution; Abcam, Tokyo, Japan), rabbit anti-CD206 (ab64693, 1:1000 dilution; Abcam), rabbit anti-IL-8 (ab106350, 1:500 dilution; Abcam), and rabbit anti-PAI-1 (ab66705, 1:1000 dilution; Abcam). Samples were washed with TBST, and 100–400 μL of DAB (Peroxidase Stain DAB Kit; Nacalai Tesque, Kyoto, Japan) was applied as a chromogen to each section. Finally, Mayer’s hemalum solution (1:4 dilution; Merck KGaA, Darmstadt, Germany) was used for counterstaining, and then sections were washed twice for 5 min each in dH2O. After dehydration, sections were mounted with coverslips.
8
Immunofluorescence Analysis of PAI-1 and tPA in Rat Hippocampus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The rat brain samples were fixed (4% paraformaldehyde, PFA), gradient sugar deposition (10, 20, 30% sucrose), and then coronal sections (35 mm in thickness) of the Hip were sliced using a freezing microtome (CM 1850; Leica, Germany). Sections were incubated in rabbit anti-PAI-1 (ab66705, Abcam, United Kingdom; 1:200) and rabbit anti-tPA (10147-1-AP, Proteintech, China, 1:50), 4°C overnight. After the sections were washed with 0.01 PBS, they were incubated with goat anti-mouse IgG H&L (Alexa Fluor® 488) (ab150113, Abcam, United Kingdom; 1:200) or goat anti-rabbit IgG H&L (Cy3 ®) preadsorbed (ab6939, Abcam,United Kingdom; 1:200) as secondary antibody at room temperature for 2 h. A laser scanning confocal microscope (LSM 900, Zeiss, Germany) was used to observe the positive cells.
9
Isolation and Characterization of Primary Mouse Chondrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary chondrocytes from 5 day old mice were isolated according to published protocol18 (link). Briefly, 5 day old C57bl/6 PER2:Luc mice were sacrificed by decapitation. Knee, hip and shoulder joints were dissected, and any soft tissue removed. Joint cartilage subjected to pre-digestion with collagenase D 3 mg/mL in DMEM two times for 30 min at 37°C with intermittent vortex to remove soft tissue leftovers. Subsequently, the cartilage was diced using a scalpel and digested overnight at 37°C. Cells were dispersed by pipetting and passed through 70μM cell strainer. Cell suspension was then centrifuged and the pellet was re-suspended in DMEM/F12 with 10% FBS and plated in T75 flasks. Cells were passaged only once before performing experiments.
Western blotting was performed according to standard procedures. Primary antibodies for CTGF (Abcam ab6992), PAI-1 (Abcam ab66705), MATN1 (Abcam ab106384) and alpha tubulin (Sigma T9026) were used in 1:2000 dilution. Secondary antibodies (LI-COR IRDye 800CW and 680RD) were used in 1:20,000 dilution. WB was quantified using the LI-COR Odyssey Imaging System.
Western blotting was performed according to standard procedures. Primary antibodies for CTGF (Abcam ab6992), PAI-1 (Abcam ab66705), MATN1 (Abcam ab106384) and alpha tubulin (Sigma T9026) were used in 1:2000 dilution. Secondary antibodies (LI-COR IRDye 800CW and 680RD) were used in 1:20,000 dilution. WB was quantified using the LI-COR Odyssey Imaging System.
10
Immunofluorescent Analysis of Rat Brain Tumor
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!