Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining of DNA strand breaks was performed using the In Situ Death Detection Kit (Roche, Branchburg, NJ, USA), as previously described (55 (link)). Fresh frozen heart sections were cut using a Leica cryostat (Model CM3050S, Leica Microsystems) to produce 7-µm tissue sections. Tissue sections were fixed with 4% paraformaldehyde for 20 min and permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min at 4°C. To distinguish myocytes from nonmyocytes in the heart, tissue sections were incubate with anti-Desmin (Cell Signaling, 1:100) followed by incubation with anti-Rabbit secondary conjugate with Alex Fluor 568 (Invitrogen). Fifty microliters of a reaction mixture containing terminal deoxynucleotidyl transferase (TdT), fluorescein-dUTP was added to each section and incubated in a humidified chamber for 60 min at 37°C. Sections were washed three times with PBS and counterstained with 4’,6’-diamidino-2-phenylindole (DAPI, 5 µg/ml) for 1 min. Slides were mounted with the Prolong Gold mounting medium (Invitrogen, Carlsbad, CA, USA), and five images per tissue section were obtained using a Zeiss confocal microscope equipped with an Olympus MagnaFire SP digital camera and ImagePro image analysis software as previously described (56 (link)).
Anti desmin
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-desmin is a laboratory reagent used for the detection of the desmin protein in biological samples. Desmin is an intermediate filament protein found in muscle cells. Anti-desmin is a highly specific antibody that binds to desmin and can be used in various immunoassay techniques to identify and quantify the presence of desmin in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Anti α sma, by Cell Signaling Technology (2 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Cryostat, by Leica (1 mentions)
In situ death detection kit, by Roche (1 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
Alex fluor 568, by Thermo Fisher Scientific (1 mentions)
Magnafire sp digital camera, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti pro caspase 3, by Cell Signaling Technology (1 mentions)
Anti alpha smooth muscle actin, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Anti gfap, by Cell Signaling Technology (1 mentions)
Anti phospho stat3, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
10 protocols using anti desmin
1
TUNEL Staining of DNA Strand Breaks
2
Immunofluorescence Staining of Mouse Liver and Primary Hepatic Stellate Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For in vivo experiments, fresh frozen AAV transfected mouse liver tissues were sectioned to 5 μM and stained with DAPI (Beyotime) for 10 min. For in vitro experiments, primary HSCs were first fixed in 4% paraformaldehyde for 15 min and then permeabilized in 0.5% Triton X-100 for 15 min at room temperature. Cells were then blocked by 5% BSA and incubated with anti-desmin (5332, Cell Signaling Technology, 1:100 dilution) or anti-α-SMA (19245, Cell Signaling Technology, 1:200 dilution) antibodies at 4 °C overnight followed by appropriate secondary antibodies conjugated with Alexa Fluor 647. Finally, the cell nuclei were stained with DAPI. Images were visualized and captured by using a Leica SP8 confocal microscope.
3
Protein Expression Analysis in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were homogenised and lysed in RIPA buffer supplemented with proteinase inhibitor. An equal amount of proteins (25 μg) were loaded and run on 12% SDS-PAGE gel and transferred onto PVDF membranes following electrophoresis. After the incubation with 5% milk in TBS/T for 1 h, the membrane was incubated with the primary antibodies at 4 °C overnight. The primary antibodies used in this experiment were: anti-total AKT, anti-total Erk, anti-Phospho-AKT, anti-Phospho Erk1/2, anti-procaspase 3, anti-Caspase3, anti-PI3K, anti-beta-actin, anti-GFAP, anti-vimentin, anti-desmin, anti-alpha-smooth muscle actin, and anti-Phospho Stat3 (Cell signaling, Danvers, MA, USA). GAPDH (Cell Signaling) was used as the loading control.
4
Pericyte Phenotype and CXCL3 Secretion
5
In Vitro Cardiac Cell Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher (Waltham, MA, USA). Ang II, ACh, atropine, penicillin, streptomycin, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), DMSO, and propidium iodide (PI) were obtained from Sigma-Aldrich (Saint Louis, MI, USA). Anti-Vimentin, Anti-Desmin, anti-GAPDH, anti-TGF-β1, anti-Smad3, anti-P-Smad3, and anti-MMP-2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Von Willebrand Factor, Anti-I collagen, Anti-III collagen, anti-α-SMA and fluorescein-isothiocyanate-conjugated (FITC) anti-rabbit second antibody were purchased from Abcam (Cambridge, UK). 680RD Conjugated Goat polyclonal Anti-Mouse IgG and 800CW Conjugated Goat polyclonal Anti-Rabbit IgG were purchased from LiCor Biosciences (Lincoln, NE, USA). Collagenase II was from Worthington (Columbia, NJ, USA). PYR was obtained from Shanghai ZhongxiSunve Pharmaceutical (Co, Ltd, Shanghai, China).
6
Immunofluorescent Staining of Oligomers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Indirect immunofluorescent staining was performed as previously described [10 (link)]. Primary and secondary antibodies used in this study include anti-pre-amyloid oligomers (Millipore, #AB9234), anti-desmin (Cell Signaling, #5332), alexa-fluor-594 goat anti-rabbit IgG (Invitrogen, #A11012), and alexa-fluor-488 goat anti-mouse IgG (Invitrogen, #A11001). Following secondary antibody incubation and PBS wash, coverslips were mounted using DAPI-mounting solution (VectaShield, #H1200).
7
Immunofluorescence Staining of Kidney
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immuno uorescence staining of kidney tissues was performed on 5 µm thick frozen sections. Brie y, sections were xed in methanol for 10 minutes, blocked with 5% BSA for 1 hour and then blocked with anti-USP25 (Santa Cruz, sc-398414, 1:50), anti-Desmin (Cell Signaling Technology, #5332, 1:200), and anti-SMAD2 (Cell Signaling Technology, #5339, 1:200) overnight. The uorescence-conjugated secondary antibody (YEASEN) was then incubated at 37°C for 1 hour. Sections were counterstained with 4',6diamidino-2-phenylindole (DAPI) and imaged by Nikon microscope imaging system (Nikon Instruments, Japan).
8
Western Blot Analysis of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-cTnT, anti-β-actin and anti-Ki67 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-GATA4 and anti-desmin were bought from Cell Signaling Technology (Danvers, MA, USA). Anti-SMO, anti-Bax and anti-Bcl-2 antibodies were purchased from Abcam (Cambridge, MA, USA). Cells were added into tubes containing lysis buffer (50 mmol/l Tris-HCl, 0.2% sodium deoxycholate, 0.2% SDS, 1% Triton X-100, and 1 mmol/l EDTA at pH 7.4). The lysate supernatant was collected after centrifugation at 14,000 g for 30 min at 4°C. Subsequently, protein concentrations were determined using a BCA protein detection kit (Keygen Biotech. Co. Ltd., Nanjing, China) according to the manufacturer's instructions. Western blot assay was performed as previously described (Shen et al., 2012 (link)).
9
Protein Expression Analysis in Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins from tissues or cells were extracted by lysing in RIPA lysis buffer containing a protease inhibitor (Applygen, Beijing, China). The proteins were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (MilliporeSigma, Burlington, MA, USA). After blocking with Tris-buffered saline containing 0.05% Tween-20 (TBST) and 5% non-fat dry milk or 5% BSA for 1 h at room temperature, the membranes were incubated with anti-α-SMA (1:1000; #19,245; Cell Signaling Technology, Danvers, MA, USA), anti-desmin (1:1000; #5332; Cell Signaling Technology), anti-PCNA (1:500; sc-56; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-β-actin (1:1000; sc-47778; Santa Cruz Biotechnology, Inc.) antibodies at 4 °C overnight, followed by incubation with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at 25 °C. β-actin was used as an internal control. Proteins were detected with ECL chemiluminescence and their intensity was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
10
Immunoblot Analysis of Pannexin Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HSMM, SkMC, and HEK293T cell lysates were obtained as previously described (7 (link)). After separation by SDS-PAGE, proteins were transferred to PVDF membranes and immunoblotted with anti-Panx1 (3 (link)), anti-Panx2 (28 (link)), anti-Panx3 (3 (link)), anti-desmin (Cell Signaling, Whitby, ON, Canada), anti-MHC (R&D Systems, Minneapolis, MN), and anti-PCNA (Dako, Burlington, ON, Canada). Alexa 680 (Invitrogen) or infrared fluorescent-labeled secondary antibodies (1:5000) (IRDye 800 (Rockland Immunochemicals, Gilbertsville, PA)) were used, and immunoblots were quantified using the Odyssey infrared-imaging system (Licor). The membranes were reprobed for tubulin for normalization of protein loading. Protein standards are depicted in kDa. The relative intensities of the bands represent the average ± S.D. of at least three independent Western blots. Lysates from HEK293T cells overexpressing either Panx1, Panx2, or Panx3 have been used as positive controls.
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!