After dewaxing with xylene, kidney paraffin sections were immersed in gradient alcohol and water before staining, then soaked in 3% H2O2 to remove endogenous peroxidase. All sections were heated in sodium citrate solution for 15 min in 98°C for antigen retrieval. Then the tissue specimens were circled with an immunohistochemical pen, added 5% bovine serum albumin (BSA) solution, and blocked at 37°C for 30 min, incubated with the primary antibodies including fibronectin (1:500, abcam, ab268021, United Kingdom, Cambridge), alpha smooth muscle actin (1:300, abcam, ab7817, United Kingdom Cambridge), collegen-I (1:500, abcam, ab270993, United Kingdom, Cambridge) solution 200 μL at 4°C overnight. After washing off, these tissues were incubated with secondary antibody solution at 37°C for 40 min, colored by 3,3′-diaminobenzidine (DAB) under a microscope and counterstained with hematoxylin (Cat. No. C0105S, Beyotime Biotechnology, Shanghai, China). The immunohistochemistry kits were purchased from Boster Biotechnology (Fujian, China). DAB color reagent kits were purchased from Maixin Biotechnology (Fujian, China).
Ab270993
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab270993 is a laboratory product offered by Abcam. It serves as a tool for researchers in their scientific investigations. The core function of this product is to facilitate specific experimental procedures, though details about its intended use are not provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab7778, by Abcam (8 mentions)
Ab8226, by Abcam (7 mentions)
Ab124964, by Abcam (6 mentions)
Ab8245, by Abcam (5 mentions)
Ab184993, by Abcam (5 mentions)
Ab5694, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ab236639, by Abcam (3 mentions)
Ab32572, by Abcam (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Ab2413, by Abcam (3 mentions)
Ab92547, by Abcam (3 mentions)
Ab232784, by Abcam (3 mentions)
Ab7817, by Abcam (2 mentions)
Ab254360, by Abcam (2 mentions)
Ab196495, by Abcam (2 mentions)
Ab288151, by Abcam (2 mentions)
Ab32503, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Ab214821, by Abcam (2 mentions)
50 protocols using ab270993
1
Kidney Tissue Immunohistochemistry Analysis
2
Immunohistochemical Analysis of ECM Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunohistochemical analyses, the sections were exposed overnight at 4°C to antibodies against E-cadherin (rabbit monoclonal; 1 : 200; ab76319; Abcam), COL I (rabbit monoclonal; 1 : 200; ab270993; Abcam, Cambridge, UK), MMP-9 (rabbit monoclonal; 1 : 200; ab76003; Abcam), TIMP-1 (rabbit polyclonal; 1 : 200; Abcam), α-SMA (mouse monoclonal; 1 : 200; ab7817; Abcam), or fibronectin (rabbit monoclonal; 1 : 200; ab199056; Abcam), followed by incubation with secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1 : 500; 115-035-003; Jackson ImmunoResearch, Ely, UK).
3
Immunohistochemical Analysis of Rat Femur Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Harvested rat femurs were fixed in 4% paraformaldehyde (PFA) then decalcified in ethylenediaminetetraacetic acid (EDTA; 10%, pH 7.0) prior to embedding in paraffin. Samples were sliced into 5 μm-thick sections using a microtome in a plane parallel to that of the bone defect cavity. The sections were then incubated in dimethylbenzene I and II, twice in 100% ethanol, twice in 95% ethanol, then in 90% ethanol and 80% ethanol, respectively. Endogenous peroxidase activity was eliminated with 3% H2O2 after which samples were incubated with goat serum at room temperature for 30 min. Finally, the samples were incubated with a specific primary antibody for 12 h at 4 °C, from the following panel: mouse monoclonal anti-collagen I (Col-1;ab270993, Abcam); rabbit polyclonal anti-CD29 (GeneTex,California,USA,GTX128839); rabbit anti-mouse Sca-1 (Biolegend, California,USA,108,103) and Rabbit IgG(ab125938) which was regarded as negative control in IHC. Sections were then incubated with a biotinylated secondary antibody or conjugated with horseradish peroxidase (HRP) at room temperature for 30 min, after which color was developed with diaminobenzidine (DAB) and the sections counterstained with hematoxylin. Images were acquired following observation by light microscopy (Olympus BX53, Tokyo, Japan).
4
Histological Analysis of Supraspinatus Tendon
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples used for histological analysis were routinely fixed in 10% neutral buffered formalin, decalcified with 10% EDTA, embedded in paraffin, and cut into 4 μm-thick sections.
To obtain an overview of the supraspinatus tendon, the sections were stained with H&E. To detect the specific expression levels of type I collagen and type III collagen, the sections were stained with collagen I antibody (1:500, Abcam, ab270993) and collagen III antibody (1:400, Abcam, ab6310).
To obtain an overview of the supraspinatus tendon, the sections were stained with H&E. To detect the specific expression levels of type I collagen and type III collagen, the sections were stained with collagen I antibody (1:500, Abcam, ab270993) and collagen III antibody (1:400, Abcam, ab6310).
5
Western Blot Analysis of Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The target cell protein was obtained by RIPA method, and the protein concentration was determined by BCA kit (Solarbio, Beijing, China). Then, prepared protein was taken for gel electrophoresis separation, and the isolated protein was electrically transferred to PVDF membrane. After blocked with 5% skim milk, the PVDF membrane was subjected to primary antibodies: connective tissue growth factor (CTGF, #ab6992, 1:1000, Abcam, Cambridge, UK), collagen I (#ab270993, 1:1000, Abcam, Cambridge, UK), collagen III (#ab7778, 1:1000, Abcam, Cambridge, UK), Bcl-2 (#ab196495, 1:1000, Abcam, Cambridge, UK), Bax (#ab32503, 1:1000, Abcam, Cambridge, UK), NOD-like receptor protein 3 (NLRP3, #ab263899, 1:1000, Abcam, Cambridge, UK), ASC (rat: #ab180799, 1:1000, Abcam, Cambridge, UK; mouse: #67824S, Cell Signaling, Danvers, MA), caspase-1 (#83383, 1:1000, Cell Signaling, Danvers, MA), IL-β (#ab254360, 1:1000, Abcam, Cambridge, UK), IL-18 (#ab191860, 1:1000, Abcam, Cambridge, UK) and GAPDH (#ab9485, 1:1000, Abcam, Cambridge, UK). The next day, the PVDF membrane was incubated with secondary antibody (#ab288151, 1:5000, Abcam, Cambridge, UK) at room temperature. An enhanced chemiluminescence kit (ECL, #P0018S, Beyotime Biotechnology, Beijing, China) was added and exposed in the gel imaging system. The protein content was analysed using Quantity-One software (Bio-Rad, Hercules, CA).
6
Protein Analysis of Bone Tissues and Osteoblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total proteins of bone tissues and osteoblasts was extracted, and the protein content was measured by BCA assay kit. The proteins were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the polyvinylidene fluoride (PVDF) membrane. After the end of the film transfer, each blot were blocked with 5% skim milk for 1 h. Then the primary antibody was added and incubated at 4°C overnight. After the incubation membrane was washed, the secondary antibody was added. ECL kit was used for photoluminescence development, and GAPDH (ab8245, abcam, United States, 1:1,000) was used as the reference protein. The grayscale values of each band were analyzed by ImageJ software. The information of primary antibodies was listed as follows: β-catenin (ab32572, abcam, United States, 1:5,000), TCF (ab185736, abcam, United States, 1:1,000), LEF-1 (ab137872, abcam, United States, 1:1,000), cyclinD (#2978, Cell Signaling Technology, United States, 1:1,500), c-myc (ab32072, abcam, Unites States, 1:1,000), Runx2 (ab236639, abcam, United States, 1:1,000), COL1A1 (ab270993, abcam, United States, 1:1,000), BMP-2 (ab214821, Abcam, United States, 1:1,000), OPN (ab228748, abcam, United States, 1:1,000).
7
Collagen-I Immunofluorescence of Intestinal Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The intestinal tissue samples were cut into the pieces of 5mm × 5 mm and fixed overnight in 4 % Paraformaldehyde (PFA) fix solution. The fixed intestinal tissues were embedded in paraffin, and cut into sequential 8-μm thick slides. The standard protocol of the immunofluorescent staining was performed according to the report [22 ]. Collagen-I (1:100, ab270993, Abcam, USA) staining area and extent were assessed under microscope examination. The quantitative analyses was performed by using the software (LAS X Core Software, Leica Microsystems, GER).
8
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein samples were extracted from cell samples with RIPA buffer (CWBio, Beijing, China) and protein concentration was measured using BCA protein assay kit (Beyotime, Shanghai, China). Equal amount of protein sample was separated on 12% SDS-PAGE gels followed by transferred to nitrocellulose membranes. Then, the membranes were blocked in 5% non-fat milk dissolved in TBST solution and incubated with primary antibodies against p300 (Abcam, Cambridge, MA, USA; ab275378, 1:1000 diluted), RhoA (ab187027, 1:2000 diluted), ROCK (ab134181, 1:1000 diluted), NF-κB p65(ab207297, 1:1000 diluted), HDAC1 (ab109411, 1:4000 diluted), COL1A1 (ab270993, 1:3000 diluted), COL2A1 (ab34712, 1:3000 diluted), and GAPDH (ab8245, 1:5000 diluted) overnight at 4°C. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 2 h. The protein bands were visualized using an enhanced chemiluminescence reagent (Pierce Biotech, Inc., Rockford, IL, USA).
9
Immunohistochemical Analysis of Kidney Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 4-μm sections were rehydrated, blocked with 3% H2O2 and placed in a microwave for antigen retrieval. Then, the sections were blocked in 5% bovine serum albumin for 1 h and hybridized with the primary antibodies at 4°C overnight, followed by the incubation with secondary antibody at 37°C for 45 min. Phosphate-buffered saline (PBS) was used instead of primary antibody as negative control. 3,3ʹ-diaminobenzidine (DAB) was used for color development, and the nuclei were counter-stained with hematoxylin. The staining was observed under the microscope. The positive cells were stained in dark brown. The optical density (OD) in each section was evaluated using the Image J. The primary sections used were Podocin (1:500, ab181143, Abcam Inc., Cambridge, MA, USA), FOXA1 (1:1000, ab170933, Abcam), SATB1 (1:100, ab109122, Abcam), β-catenin (1:5000, ab32572, Abcam), Collagen I (1:500, ab270993, Abcam), Fibronectin (1:100, ab2413, Abcam) and transcription factor 4 (TCF4; 1:50, MA5-35295, Thermo Fisher Scientific). The secondary antibody used was IgG H&L (HRP) (1:2000, ab205718, Abcam).
10
Immunohistochemical Analysis of Liver Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver tissues were fixed in 4% paraformaldehyde and were subsequently embedded in paraffin. IHC staining was performed on 5 μm slices and stained using the protocol as described previously [24 (link)]. In brief, the sections were deparaffinized in xylene and rehydrated in graded alcohols, followed by blocking the endogenous peroxidase activity in 0.3% hydrogen peroxide. The sections were then incubated with primary antibodies against collagen I (1:100, ab270993, Abcam), α-SMA (1:200, ab124964, Abcam), METTL3 (1:100, ab195352, Abcam), and Sp1 (1:500, SAB4502837, Sigma-Aldrich) at 4 °C overnight, followed by the incubation with the biotinylated anti-rabbit secondary antibody (1:1000, ab6720, Abcam) for 1 h at room temperature. Afterward, the avidin peroxidase conjugate (1:2000, ab59653, Abcam) was added for 1 h at room temperature. Finally, the target signal was developed utilizing DAB substrate (Vector Labs), and the slices were stained with hematoxylin. The images were taken under a light microscope (Zeiss, Germany). The measuring process was conducted by an assessor blind to treatment allocation.
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!