Following four weeks of consistent hyperglycemia, both hyperglycemic and age-matched control mice were anesthetized with isoflurane (Baxter, Deerfield, IL) and an area (~10 cm2) of the skin just posterior to the skull was denuded by clippers then swabbed with Betadine® (Purdue Fredrick Co., Norwalk, CT) and 70% ethanol three times. Four 4 mm punch biopsies were performed on the shaved area utilizing sterile single-use biopsy punches. Wound healing was assessed daily for fifteen days by measuring wound closure from photographs using ImageJ (NIH Image; https://rsbweb.nih.gov/ij/ ). For comparison, a 4 mm circular paper biopsy reference was included in each wound photograph as a means of standardizing photos. Wound tissue was collected on days 0, 1, 3, 7, and 10 for RNA, protein, and histological analysis. Wound tissue was immediately homogenized in TRI Reagent (Molecular Resource Center, Cincinnati, OH) for RNA or RIPA buffer plus PMSF and protease inhibitor (cat. no. P8340; Sigma) for protein isolation or fixed in 10% buffered formalin for histology. Sections of these samples were stained with hematoxylin/eosin or stained for SOCS3 (ab76315, Abcam, Cambridge, UK) and pSTAT3 (ab76315, Abcam) followed by anti-rabbit Alexa Fluor-488 (Life Technologies, Carlsbad, CA) and nuclear staining with DAPI.
Ab76315
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab76315 is a laboratory equipment product offered by Abcam. It is a device designed for specific laboratory functions. The core function of this product is to facilitate laboratory procedures, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab68153, by Abcam (61 mentions)
Ab108596, by Abcam (27 mentions)
Ab32101, by Abcam (25 mentions)
Ab119352, by Abcam (24 mentions)
Pvdf membrane, by Merck Group (17 mentions)
Ab181602, by Abcam (10 mentions)
Ab8245, by Abcam (9 mentions)
Bca kit, by Beyotime (9 mentions)
Ab205718, by Abcam (8 mentions)
Ab32503, by Abcam (8 mentions)
Ab9485, by Abcam (8 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Ab8226, by Abcam (7 mentions)
Ab133666, by Abcam (7 mentions)
Ab138005, by Abcam (6 mentions)
Ripa lysis buffer, by Beyotime (6 mentions)
Ab8227, by Abcam (5 mentions)
Ripa buffer, by Beyotime (5 mentions)
Ab109085, by Abcam (5 mentions)
Bca protein assay kit, by Beyotime (5 mentions)
168 protocols using ab76315
1
Wound Healing Dynamics in Hyperglycemic Mice
2
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from HUVECs using radio-immunoprecipitation assay cell lysis (R0010; Solarbio) and refined using a bicinchoninic acid protein assay kit (GBCBIO Technologies, Guangzhou, Guangdong, China). The proteins (40 μg) were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto the polyvinylidene fluoride membrane (Merck Millipore, Billerica, MA, USA). The membranes were probed with primary rabbit antibodies as follows: β-actin (1:1,000, Rabbit, ab8227, Abcam, Cambridge, UK), EZH2 (# 5246, 1:1,000, Rabbit, CST, USA), phosphorylated-STAT3 (p-STAT3) (1:2,000, Rabbit, ab76315, Abcam), STAT3 (1:1,000, Rabbit, ab68153, Abcam), cleaved-caspase3 (#9661, 1:1,000, Rabbit, CST), and cleaved-poly ADP-ribose polymerase (cleaved-PARP) (#5625, 1:1,000, Rabbit, CST). The results were visualized using horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) (ab205718, 1:2,000, Abcam) and enhanced chemiluminescence detection reagents. Image J software was used to analyze relative protein expression by analyzing the ratio of intensity of protein bands to be tested against that of β-actin.
3
Immunohistochemical Staining Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The paraffin-embedded sections were treated with immunohistochemical staining using the primary antibody EZH2 (#5246, 1:50, Rabbit, CST), HMGB1 (1:250, Rabbit, ab79823, Abcam), p-STAT3 (1:500, Rabbit, ab76315, Abcam), cleaved-caspase3 (#9661, 1:200, Rabbit, CST), and cleaved-PARP (#5625, 1:150, Rabbit, CST). The samples were observed under a Leica microscope (Leica M205C). Positive stains were scored according to a previously published method (20 (link)).
4
Western Blot Analysis of Stem Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The in vitro-cultured SSCs were collected, and protein lysates (keygentec, Nangjing, China) were extracted at 4 °C for 30 min. The proteins were denatured in 5× SDS loading buffer at 100 °C for 5 min. The total cell proteins were resolved by 12% SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% nonfat milk powder in TBST for 1 h, the PVDF membranes were incubated with polyclonal rabbit anti-Osm (1:1000, A6163, ABclonal, Woburn, MA, USA), monoclonal mouse anti-β actin (1:1000, sc-58673, Santa Cruz Biotechnology), polyclonal rabbit anti-Socs3 (1:1000, 14025-1-AP, Proteintech, Wuhan, China), monoclonal rabbit anti-p-Stat3 (1:1000, ab76315, Abcam), polyclonal rabbit anti-p-Akt (1:1000, AF0016, AFFinify) and polyclonal rabbit anti-GAPDH (1:1000, bs2188R, Bioss, Beijing, China) primary antibodies at 4 °C overnight. Then, horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit(1:20000, A21020-1, Abbkine) secondary antibodies were used at room temperature for 1 h, and the protein expression was detected by enhanced chemiluminescence (RM00021, ABclonal); gel imaging was performed with a ChemiDoc™ XRS+ (Bio-Rad, USA) with Image Lab™ software, and gray value analysis was performed with ImageJ.
5
Signaling Pathway Modulation in HSFs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HSFs were cultured in 100 mm Petri dishes. The inhibitor group was first treated with the small-molecule inhibitors SB431542, LY294002, AG490 or PD98059 for 2 h (10 μM). The inhibitors SB203583 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (an ERK1/2 inhibitor), AG490 (a JAK inhibitor) and BX795 (a TBK1 inhibitor) were purchased from Millipore (Billerica, MA). Then, the cells were treated with 3D-GF-PADM at 1 mg ml−1 for 24 h. After collection, total protein extraction was done with RIPA buffer containing phosphatase and protease inhibitors (Pierce, Rockford, IL, USA). After separation using a 12% SDS-PAGE denaturing gel and transfer to PVDF membranes, the membranes were blocked with 5% skim milk. The primary antibodies, including JAK2 (Abcam; ab108596), p-JAK2 (Abcam; ab32101), STAT3 (Abcam; ab68153), p-STAT3 (Abcam; ab76315) and HAS2 (Abcam; ab140671), were used at 4 °C overnight. Secondary antibody incubation was performed at room temperature for 1 h.
6
Nerve Regeneration Protein Analysis
7
Investigating Protein Interactions in U251 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the interaction between Aβ and CAV2, TGFB2 or TGFBR1, U251 cells were inoculated with β-amyloid (1–42), HiLyte Fluor™ 488-labelled (ANASPEC, AS-60479-01). After 1 h, the cells were washed, fixed and incubated with CAV2 (Abcam, ab133484), TGFB2 (Abcam, ab36495) or TGFBR1 antibodies (Abcam, ab31013). The cells were washed, counterstained with DAPI and observed under an Olympus FV1000 confocal laser microscope. To study the interaction between NEAT1 and P300/CBP, U251 cells were incubated with the NEAT1 probe overnight at 37 °C and then the anti-P300 (Abcam, ab59240) or anti-CBP antibodies (Abcam, ab50702) for 1.5 h at room temperature. After the cells were washed and incubated with the secondary antibody, they were counterstained with DAPI and mounted for observation. Cell images were obtained with an Olympus FV1000 confocal microscope. To study the role of NEAT1 in the interaction between STAT3 and H3K27Ac, U251 cells were transfected with a NEAT1 or negative control siRNA for 36 h and then incubated with the anti-STAT3 Y705 (Abcam, ab76315) and anti-H3K27Ac antibodies (Abcam, ab4729) for 1.5 h at room temperature. After the cells were washed and incubated with the secondary antibody, they were counterstained with DAPI and mounted for observation. Cell images were obtained with an Olympus FV1000 confocal microscope.
8
Western Blot Analysis of STAT3, c-Met, and PD-L1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted with lysis buffer (Beyotime Biotechnology, China), separated by 10 % polyacrylamide gel electrophoresis (PAGE), and subsequently transferred to a nitrocellulose membrane (Beyotime Biotechnology, China) using a wet transfer apparatus. After blocking the samples with 5 % bovine serum albumin for 30 min at room temperature, they were incubated overnight at 4 °C with the following primary antibodies: anti-total STAT3 (ab68153), anti-phosphorylated STAT3 (ab76315), anti-c-Met (ab216334), and anti-PD-L1 (ab205921), which were all obtained from Abcam, UK. Subsequently, the membrane was incubated with secondary antibodies for 2 h and visualized using a gel imaging system (Thermo Fisher, USA).
9
Western Blot Analysis of Cancer Biomarkers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
This study utilized RIPA buffer (Beyotime Institute of Biotechnology) that contained protease K inhibitor for extracting total proteins, which were then separated through 12% SDS-PAGE, followed by transfer onto PVDF membrane. Subsequently, membrane was immersed within 5% defatted milk under ambient temperature for a 2 h, followed by overnight incubation under 4 °C using corresponding primary antibodies: EZH2 (1:500, ab191080; Abcam, Cambridge, MA, USA), p-STAT3 (1:2000, ab76315; Abcam, Cambridge, MA, USA), STAT3 (1:1000, ab68153; Abcam, Cambridge, MA, USA), Mcl-1 (1:1000, ab32087; Abcam, Cambridge, MA, USA) and p-Bcl-2 (1:1000, ab218123; Abcam, Cambridge, MA, USA), H3K27me3 (1:1000, ab6002; Abcam, Cambridge, MA, USA) with GAPDH being the loading reference. Then, the membrane was subject to secondary antibody incubation (1:5000, ab96899; Abcam, Cambridge, MA, USA). Protein band visualization was performed using Chemidoc EQ system (Bio-Rad Laboratories, Inc.).
10
Protein Expression Analysis of Lung Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lung tissues and cells were homogenized and incubated in lysis buffer containing a protease inhibitor cocktail. The protein concentration was measured using a BCA kit (KGA902, KeyGEN BioTECH, Nanjing, China), and the proteins were then denatured at 100°C for 5 min. The proteins were loaded onto a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The blots were blocked with 5% nonfat milk at room temperature for 1 h and incubated with primary antibodies overnight at 4°C. The primary antibodies included rabbit monoclonal anti-Nrf2 (ab137550, Abcam, 1 : 1,000), anti-SLC7A11 (ab37185, Abcam, 1 : 1,000), anti-STAT3 (ab68153, Abcam, 1 : 2,000), anti-pSTAT3 (ab76315, Abcam, 1 : 2,000), and anti-β-actin (4970S, Cell Signaling Tech, 1 : 1,000). After washing three times with TBST for 15 min, the strips were incubated with anti-mouse or anti-rabbit horseradish peroxidase- (HRP-) conjugated secondary antibodies and detected using an ECL detector. The signals were scanned and quantified using ImageJ software.
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!