Fixed samples underwent standard processing for paraffin fixation. Serial paraffin sections were cut at 5 μm thickness. Tissue samples from in vivo implantation experiments were cut longitudinally along the whole length of the scaffold. Hematoxylin and eosin (H&E) staining was performed using an automated staining system (Tissue-Tek). hMABs were identified in murine xenograft experiments using an anti-human cell antibody, STEM121 (Clontech; Y40410; 1:1000), that identifies a cytoplasmic antigen present in human but not rodent cells, and an anti-luciferase antibody (Abcam; ab181640, 1:100). Detection was using a species-appropriate HRP-conjugated antibody, and the counterstain was hematoxylin.
Ab181640
Manufactured by Abcam
Sourced in United States
Ab181640 is an Recombinant antibody that binds to Calcium/calmodulin-dependent protein kinase II (CaMKII). CaMKII is a serine/threonine-protein kinase that is involved in the calcium-mediated signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Stem121, by Takara Bio (1 mentions)
Direct red 80, by Merck Group (1 mentions)
F3648, by Merck Group (1 mentions)
Ab19808, by Abcam (1 mentions)
V4888, by Merck Group (1 mentions)
Ab64693, by Abcam (1 mentions)
Goat on rodent, by Biocare Medical (1 mentions)
Af471, by R&D Systems (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ab260038, by Abcam (1 mentions)
Ab187340, by Abcam (1 mentions)
Ab100961, by Abcam (1 mentions)
Ab170904, by Abcam (1 mentions)
Temozolomide, by Selleck Chemicals (1 mentions)
Lapatinib, by Selleck Chemicals (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab24115, by Abcam (1 mentions)
Anti mouse igg alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Protein block, by Agilent Technologies (1 mentions)
7 protocols using ab181640
1
Histological Analysis of Xenograft Implants
2
Kidney Tissue Analysis by Histology
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Kidneys were fixed in 10% formalin and embedded in paraffin. For detection of Sirius red-positive collagen, deparaffinized kidney sections (4 μm) were rehydrated, predifferentiated with 0.2% phosphomolybdic acid for 5 min, stained with 0.1% picrosirius red (Direct Red 80, REF: 365548; Sigma-Aldrich, St. Louis, MO, USA) in picric acid for 90 min and differentiated with saturated picric acid. Primary antibodies against fibronectin (1:4800, F3648, Sigma), collagen type IV (1:500, ab19808, Abcam, Cambridge, UK), endomucin (1:20,000, 14-5851-82, EBioScience, San Diego, CA), VCAM-1 (1:800, 553330, BD Biosciences, Breda, The Netherlands), F4/80 (1:100; kindly provided by the Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands) and CD206 (1:2000, ab64693, Abcam) were used for immunohistochemistry (IHC) of paraffin-embedded tissues. Frozen sections were fixed with acetone/ethanol and immunostained with primary antibodies against VSV-G (1:2000, V4888, Sigma-Aldrich), luciferase (1:3000, ab181640, Abcam), FLT-1 (1:9000, AF471, R&D Systems) and VCAM-1 (1:800, 553330, BD Biosciences). The appropriate Envision (Dako), Impress (Vector Laboratories) or Goat-on-Rodent (BioCare) HRP-conjugated secondary reagents were used with DAB+ as the chromogen. Non-specific isotype-matched antibodies were used as a negative control.
3
Evaluating Multidrug Resistance Proteins
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Lapatinib and temozolomide were purchased from Selleckchem (Houston, TX, USA). Antibodies against P-gp (ab170904, RRID:AB_2687930), BCRP (BXP-53, ab24115, RRID:AB_447879), β-actin (ab8226, RRID:AB_306371), MRP1 (ab260038, RRID:AB_2889834), MRP4 (ab77184, RRID:AB_1523967) and firefly luciferase (ab187340, RRID:AB_2889836 (DB) and ab181640, RRID:AB_2889835 (IHC)), as well as recombinant firefly luciferase protein (ab100961), were obtained from Abcam (Cambridge, MA, USA). Antibody against P-gp (C219, 517,310, RRID:AB_564389) was purchased from MilliporeSigma (St. Louis, MO, USA). Horseradish peroxidase-conjugated secondary antibodies against rat (31,470, RRID:AB_228356), mouse (31,430, RRID:AB_228307), goat (31,402, RRID:AB_228395) and rabbit (31,460, RRID:AB_228341) IgG were purchased from Thermo Fischer Scientific (Waltham, MA, USA). PBS and DPBS were purchased from HyClone (Logan, UT, USA), and DMSO was acquired from MilliporeSigma (St. Louis, MO, USA).
4
Bioluminescent Protein-Protein Interactions
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The CDS of ZmWAKLY, ZmWAKLQ, ZmBLK1, ZmWIK, ZmRBOH4 and their gene segments were cloned into the JW771-35S-CLuc vector (cLUC) and JW772-35S-NLuc vector (nLUC), respectively, to generate fusion proteins with the C-terminal or N-terminal fragment of the luciferase gene. ZmWIK and ZmWAKL were divided into their ECD/TM and ICD, marked as ZmWIKECD,TM (1-269 aa) and ZmWIKICD (270-594 aa), ZmWAKLY/ECD,TM (1-324 aa) and ZmWAKLY/ICD (325-665 aa), ZmWAKLQ/ECD,TM (1-363 aa) and ZmWAKLQ/ICD (364-704 aa). The N-terminal domain was marked as N-ZmRBOH4 (1-377 aa). The pairs of constructs were co-infiltrated into N. benthamiana leaves using the previously reported method55 (link). Two days after inoculation, 1 mM luciferin (Promega, E1601) was sprayed onto the inoculated leaves, and the luminescence signal was measured using the Chemiluminescent Imaging System (Tanon).
The luminescence signals were analyzed using ImageJ Launcher software (National Institutes of Health) to determine their intensities, so-called the mean gray value. This assay was immediately followed by protein extraction and immunoblotting with α-LUC antibody (Abcam, ab181640), and the immunoblot bands were measured using ImageJ Launcher as well. Total protein of the infiltrated leaf tissues was extracted and detected as described above.
The luminescence signals were analyzed using ImageJ Launcher software (National Institutes of Health) to determine their intensities, so-called the mean gray value. This assay was immediately followed by protein extraction and immunoblotting with α-LUC antibody (Abcam, ab181640), and the immunoblot bands were measured using ImageJ Launcher as well. Total protein of the infiltrated leaf tissues was extracted and detected as described above.
5
Evaluating Tumor Invasion and Stemness
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H&E and immunofluorescence (IF) were performed as described 36 . Tumor local invasion was evaluated by histological assessment (H&E staining) of 120 tissue sections per group (20 sections/tumor × 6 tumors/group = 120 sections/group) by means of “yes” (+) or “no” (−). Tumor invasion rate was represented in percentage. For IF, following a phosphate‐buffered saline wash, sections were blocked with Protein Block (Dako, Carpinteria, CA), then incubated with antibodies (Abs) against CD271 or Luc (ab3125 or ab181640, Abcam, Cambridge, MA, USA), and then with Alexa Fluor 594‐anti‐mouse IgG (A21203) or Alexa Fluor 594‐anti‐goat IgG (A11055, Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained with DAPI (Sigma–Aldrich, St. Louis, MO). Isotype‐matched nonspecific Abs was used as control. Immunoblot was performed as described 37 . Membranes were probed with Abs against Oct‐4, Sox‐2, and Nanog (#2750, #4900, #4893, Cell Signaling Technology, Danvers, MA, USA) or GAPDH (sc‐25,778, Santa Cruz, Santa Cruz, CA, USA) accordingly. Auto photographs of blots were scanned by densitometer (Molecular Dynamics, Caesarea, Israel) to quantify the bands. Relative levels of protein expression (fold) are presented by setting that expressed in CD271− tumor cells as “1.”
6
Transient Expression of Maize Receptor-Like Kinases
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To generate ZmWAKLY-Myc, ZmWAKLY-GFP, ZmWAKLQ-Myc, ZmWAKLQ-GFP, ZmWIK-Myc, ZmWIKECD/TM-GFP, ZmWIKICD-GFP and ZmBLK1-Myc, the CDS or gene segments for ZmWAKL, ZmWIK and ZmBLK1 were amplified and then cloned into pSuper1300 (Myc-tag or GFP-tag). The resulting plasmids were transformed into N. benthamiana leaves and expressed for about 48 h. Total protein from the infiltrated leaf tissues was extracted as described above, and the supernatant was incubated with the α-GFP magnetic beads (MBL, D153-11) at 4 °C for 2 h. The products were analyzed and detected by immunoblotting with α-Myc antibody (ABclonal, AE010) and α-LUC antibody (Abcam, ab181640). GFP and GFP-fusion proteins were detected by immunoblotting with α-GFP antibody (ABclonal, AE012).
7
Luciferase and CD3 Immunohistochemistry
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Luciferase expression of U266 cells in FFPE tissue slides was analyzed with primary Goat anti-Human antibodies against Luciferase (AB181640, Abcam, Cambridge). A monoclonal Mouse anti-Human CD3 antibody (AB699, DAKO, Netherlands) was used to stain infiltration of NK:TCR/IL-15 cells. Secondary visualization was performed using a 3,3′-Diaminobenzidine(DAB) based standard laboratory procedure.
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