OCT-embedded frozen pancreatic tumors from KPC and KPCA−/− mice were sectioned and fixed in 4% paraformaldehyde for 10 min. The tumor sections were incubated in PBS containing 0.1% Triton X-100 for 5 min and then washed with PBS. Then, the tumor sections were blocked with 10% normal goat or donkey serum in PBS for 1 hour. Next, the tumor sections were incubated with antibodies against Sema3D (Abnova), PlxnD1 (Novus), Sema3A (Abcam), Snail1 (Abcam), NG2 (Chemicon), or AnxA2 (Cell Signaling) at a dilution of 1:25, 1:50 (Snail1), 1:300 (NG2), or 1:100 (AnxA2) in 10% normal goat or donkey serum overnight at 4°C. After the overnight incubation, the tumor sections were washed and were further incubated with FITC-conjugated goat antibodies against rabbit immunoglobulin G (IgG), FITC-conjugated goat antibodies against mouse IgG (Southern Biotechnology), or AF594-conjugated donkey antibodies against rabbit IgG (Life Technologies) at a 1:200 dilution or according to the manufacturer’s instructions (AF594) in 10% normal goat or donkey serum at room temperature for 1 hour. NG2 staining was performed according to a previously described protocol (42 (link)). The tumor sections were subsequently washed and mounted in medium containing DAPI (4′,6-diamidino-2-phenylindole) (Vector Labs) before being examined under a fluorescence microscope.
Anxa2
Manufactured by Cell Signaling Technology
Sourced in United States
ANXA2 is a protein that functions in the annexin family. It plays a role in the regulation of cellular processes such as cell division, membrane organization, and ion channel activity.
Automatically generated - may contain errors
Lab products found in correlation
Bcl2 sc 492, by Santa Cruz Biotechnology (1 mentions)
Vcp ab109240, by Abcam (1 mentions)
Docetaxel, by Cayman Chemical (1 mentions)
Hspa8, by Cell Signaling Technology (1 mentions)
Succinate assay kit, by Abcam (1 mentions)
Hyou1, by Cell Signaling Technology (1 mentions)
Hsp90ab1, by Cell Signaling Technology (1 mentions)
Ki67 antibody, by Abcam (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Phosphor akt ser473, by Cell Signaling Technology (1 mentions)
Molecular imager chemidoc xrs with image lab software version 3, by Bio-Rad (1 mentions)
Phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Hsp70, by Proteintech (1 mentions)
Proliferating cell nuclear antigen (pcna), by Proteintech (1 mentions)
Glut1, by Cell Signaling Technology (1 mentions)
Glucose and lactate assay kits, by Nanjing Jiancheng (1 mentions)
Dulbecco s modified eagle s medium dmem f 12 medium, by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (1 mentions)
6 protocols using anxa2
1
Immunofluorescence Staining of Pancreatic Tumors
2
Nuclear Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear lysates of cells were extracted using a Nuclear and Cytoplasmic Extraction Kit (Pierce) according to the manufacturer's instructions. The protein concentration of nuclear lysates were quantified using BCA Protein Assay Reagent (Pierce), and then an equal amount of protein for each sample was incubated with streptavidin-conjugated agarose resin (Pierce) at 4°C overnight. Beads were washed thoroughly with IP350 (0.3%) buffer and resuspended in reducing laemmli loading buffer, and boiled for 5 minutes at 98°C. The protein samples were separated by SDS-PAGE followed by western blotting analysis. Primary antibodies used were Hspa8 (1966-1), Pcna (2714-1), Rps3a (7944-1), Ybx1 (2397-1), Prdx1 (3688-1), Bag2 (2622-1) purchased from Epitomics (Burlingame, CA), Wwox (4045S), Eif4g2 (5169P), Mcm3 (4012S), Myc (2276S), Casp3 (9662S), Eif2α (9722S), P-Eif2α (9721S), Vav1 (2502S), CHOP (5554S) and Anxa2 (8235S) purchased from Cell Signaling Technology (Danvers, MA), Oasl1 (sc-98385), Hspa5 (sc-13968), Sp110 (sc-98365), Ncl (sc-13057), and Bcl2 (sc-492) purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX), FLAG (F1804-200ug, Sigma, Saint Louis, MO), Vcp (Ab109240, Abcam Cambridge, MA), Atp2a2 (AB54032-25ul, Sangon, Shanghai, China), and Actin (TransGen Biotech Co., Ltd, Beijing, China). Secondary antibodies were purchased from Beyotime (Beyotime, Jiangsu, China).
3
Characterization of Prostate Cancer Cell Lines
4
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were prepared and analysed by western blots according to standard protocols as described previously [51 (link)] using primary antibodies, including ERK1/2 (1:2000, Cell Signaling Technology), phospho-p44/42 MAPK [ERK1/2(Thr202/Tyr204), 1:2000, Cell Signaling Technology], Akt (1:2000, Cell Signaling Technology), phosphor-Akt(Ser473) (1:2000, Cell Signaling Technology), and ANXA2 (1:2000, Cell Signaling Technology). Bands were detected using the Molecular Imager ChemiDoc XRS + with Image Lab Software version 3.0 (Bio-Rad, Hercules, CA, USA).
5
Proteomic Analysis of Cancer Stem Cells
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)/F12 medium, fetal bovine serum (FBS), and antibiotics were purchased from Gibco/BRL (Gaithersburg, MD, USA). All reagents used for two-dimensional electrophoresis (2-DE) were obtained from Bio-Rad Laboratories (Milan, Italy), and silver staining kits were obtained from CWBIO (Beijing, China). Glucose and lactate assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The cell cycle analysis kit, Dil red fluorescence cell linker kit, and ATP assay kit were purchased from Beyotime Biotech (Nantong, China). Primary antibodies against Tsg101, CD133, Nestin, GFAP, PKLR, ANXA2, ENO1, Glut-1, HK-2, and PKM-2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against HSP70, CD9, MMP-2, MMP-9, PCNA, C-myc, and GAPDH were obtained from Proteintech Group Inc. (Chicago, IL, USA). The anti-TAGLN antibody was sourced from Abcam (UK). All other reagents were obtained from Sigma-Aldrich, unless otherwise indicated.
6
Protein and Cellular Analyses of Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed by incubation on ice for 20 min. Proteins were extracted with a protein extraction kit (Beyotime, Shanghai) and used for WB assays. Tissue samples were incubated with 10% neutral buffer and then embedded in formalin paraffin for IHC. Antibodies against alpha-SMA, FAP, Ki67, E-cadherin, Vimentin, LASP1, Anxa2, p-STAT3, STAT3, Nanog, CD9, CD68, Tsg101, Bcl-2 and Bax were purchased from Cell Signaling Technology (Danvers, MA, USA). An anti-beta-actin antibody was used as the control. For IF staining, cells were seeded on previously prepared coverslips and incubated for 24 hours. After being fixed with 4% paraformaldehyde, the cells were treated with 0.1% Triton-100 and then incubated with 5% goat serum. Then, the cells were stained with primary and secondary antibodies. Primary antibodies against Vimentin, alpha-SMA and FAP were purchased from Cell Signaling Technology (Danvers, MA, USA). The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Then, fluorescence was detected using a Nikon Eclipse 80i microscope.
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!