The CT26 tumor-bearing mice with tumor volume of 150–200 mm3 were classified: Vehicle, GGd-NCPs, aAGd-NWs ([Gd] = 15.7 mg kg−1, [ara-AMP] = 34.7 mg kg−1) with or without X-ray irradiation (5 Gy × 1). The corresponding drugs were administered intravenously 6 h before radiotherapy. Then, tumor tissues were collected for evaluation of γ-H2Aχ 24 h post irradiation. Tumor tissues were sliced and stained with γ-H2Aχ mouse monoclonal primary antibody (diluted 1:200 with 3% BSA, Abcam, UK) and secondary antibody conjugated to Alexa Fluor 488 (Bioss, China) to detect DNA double-strand breaks. According to the manufacturer’s protocol, tumors were collected from six groups at 48 h post different treatments for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and Ki67 staining, respectively.
Alexa fluor 488
Manufactured by Bioss Antibodies
Sourced in China, United States
Alexa Fluor 488 is a fluorescent dye designed for use in biological applications. It has an excitation maximum at 488 nm and an emission maximum at 519 nm, making it compatible with common fluorescence detection systems. Alexa Fluor 488 is a bright and photostable dye that can be used to label a variety of biomolecules, including proteins, nucleic acids, and small molecules.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Abcam (1 mentions)
γ h2aχ mouse monoclonal primary antibody, by Abcam (1 mentions)
Mccoy 5a medium, by Merck Group (1 mentions)
Anti cav 1 antibody, by Abcam (1 mentions)
Alexa fluor 647, by Bioss Antibodies (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Cell counting kit 8 (cck8), by Beyotime (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (1 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (1 mentions)
Protease and phosphatase inhibitor cocktail, by Beyotime (1 mentions)
Hrp labeled goat anti rabbit igg h l, by Beyotime (1 mentions)
Gapdh rabbit monoclonal antibody, by Beyotime (1 mentions)
Mouse igg1 kappa isotype control, by Thermo Fisher Scientific (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Tween 20, by Solarbio (1 mentions)
Antifade mounting medium with dapi, by Beyotime (1 mentions)
Tcs sp2 clsm, by Leica (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
8 protocols using alexa fluor 488
1
Combination Radiotherapy and Nanoparticle Therapy
2
Nab-paclitaxel and Anti-HER2 Combination Therapy
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Nab-paclitaxel (Nab, Paclitaxel for Injection (Albumin Bound), provided by CSPC Pharmaceutical, China) was dissolved in 0.9% saline, Tras (Herceptin, provided by Roche Genentech, US) was dissolved in distilled water. Lova (Targetmol, China) was dissolved in DMSO, and Lova sodium (Glpbio, US) was dissolved in ethanol for preparation. RPMI-1640 medium, penicillin (100 U/ml)–streptomycin (100 mg/ml), trypsin-EDTA (0.25%) were purchased from Macgene technologies (China). McCoy 5a medium was purchased from Sigma. Cell counting kit-8, BCA protein assay kit, RIPA lysis buffer, Protease and phosphatase inhibitor cocktail, GAPDH Rabbit Monoclonal Antibody, HER2/ErbB rabbit polyclonal antibody, HRP-labeled Goat Anti-Rabbit IgG(H + L), Annexin V-FITC apoptosis detection kit, cell cycle, and apoptosis analysis kit were purchased from Beyotime technologies (China). Octyl-b-d -glucopyranoside was purchased from Millipore. Anti-Cav-1 antibody was purchased from Abcam. Alexa Fluor 488 and Alexa Fluor 647 secondary antibodies were purchased from Bioss. For transient transfection for Cav-1 knocking down and overexpression, lipofectamine 2000 was purchased from Thermo Fisher, genOFF h-CAV1_1999A was purchased from Ribobio technologies (China), and CAV1_pcDNA3.1(+) were purchased from Genscript technologies (China).
3
Colocalization of CD71 and HFn-FITC in C666-1 Cells
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Fluorescence microscopy analysis
was used to show the colocalization of CD71 and HFn-FITC within CD71-positive
C666-1 cells. Upon reaching 90% confluence, C666-1 cells were detached,
washed, and then fixed with 4% paraformaldehyde for 15 min at 37 °C.
After washing and suspending in phosphate-buffered saline (PBS), a
20 μL aliquot of the cell suspension was placed onto a slide,
allowed to sit for 20 min at room temperature, and then dried in an
oven at 37 °C. The cell smears were permeabilized with 0.1% Triton
X-100 for 15 min and blocked with 3% bovine serum albumin (BSA) for
1 h at room temperature. The cells were then incubated with the following
reagents: 5 μg/mL of mouse IgG1 kappa isotype control (Invitrogen,
USA), 5 μg/mL of CD71 mouse monoclonal antibody (Invitrogen,
USA), and 10 μg/mL of HFn-FITC in 0.1% BSA at 4 °C overnight.
Subsequently, the cells were labeled with Alexa Fluor 488 and Alexa
Fluor 555 secondary goat antimouse antibody (Bioss, China) at a dilution
of 1:100 for 1 h at room temperature. After rinsing with PBS containing
0.05% tween 20 (Solarbio, China), an antifade mounting medium with
DAPI (Beyotime Biotechnology, China) was applied to stain the cell
nuclei for 10 min. Finally, the cell smears were examined using a
confocal laser scanning microscope with 405, 488, and 543 nm wavelengths
of laser (Zeiss LSM880, Carl Zeiss AG, Oberkochen, Germany).
was used to show the colocalization of CD71 and HFn-FITC within CD71-positive
C666-1 cells. Upon reaching 90% confluence, C666-1 cells were detached,
washed, and then fixed with 4% paraformaldehyde for 15 min at 37 °C.
After washing and suspending in phosphate-buffered saline (PBS), a
20 μL aliquot of the cell suspension was placed onto a slide,
allowed to sit for 20 min at room temperature, and then dried in an
oven at 37 °C. The cell smears were permeabilized with 0.1% Triton
X-100 for 15 min and blocked with 3% bovine serum albumin (BSA) for
1 h at room temperature. The cells were then incubated with the following
reagents: 5 μg/mL of mouse IgG1 kappa isotype control (Invitrogen,
USA), 5 μg/mL of CD71 mouse monoclonal antibody (Invitrogen,
USA), and 10 μg/mL of HFn-FITC in 0.1% BSA at 4 °C overnight.
Subsequently, the cells were labeled with Alexa Fluor 488 and Alexa
Fluor 555 secondary goat antimouse antibody (Bioss, China) at a dilution
of 1:100 for 1 h at room temperature. After rinsing with PBS containing
0.05% tween 20 (Solarbio, China), an antifade mounting medium with
DAPI (Beyotime Biotechnology, China) was applied to stain the cell
nuclei for 10 min. Finally, the cell smears were examined using a
confocal laser scanning microscope with 405, 488, and 543 nm wavelengths
of laser (Zeiss LSM880, Carl Zeiss AG, Oberkochen, Germany).
4
Immunofluorescence Analysis of Co-Cultured Cells
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Co-cultures on the chamber culture slides of MalaEx with keratinocytes were fixed in cold acetone for 5 min and with monocytes in formaldehyde for 15 min. Keratinocytes were stained by a mouse anti-36/E-Cadherin (BD Biosciences), followed by a secondary anti-mouse IgG antibody conjugated with FITC (BD Biosciences). Monocytes co-cultured with MalaEx were stained with anti-CD14 conjugated with Alexa fluor 488 (Bioss Antibodies Inc., MA, USA). Glass slides were mounted with Vectashield mounting medium (Vector Laboratories, CA, USA) and fluorescent images as z-scans were acquired on a CLSM (TCS SP2; Leica Microsystems, Mannheim, Germany).
5
Immunofluorescence Analysis of Autophagy Markers
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SH-SY5Y cells were seeded on slides in 12-well culture plates. After drug treatment, the cells were fixed with ice-cold methanol for 5 min, blocked with 1% bovine serum albumin (BSA) for 30 min, and then incubated with primary antibodies against LC3 (1:200) and P62 (1:100) at 4°C overnight. The next day, the slides were incubated with Alexa Fluor 488 (Bioss Antibodies, bs-0295G-AF488, Beijing, China) or Alexa Fluor 555 (Bioss Antibodies, bs-0296G-AF555, Beijing, China) secondary antibodies (1:500) at 37°C for 1 h and incubated with DAPI (Boster, AR1177, Wuhan, China) for 5 min. Fluorescence was observed with a fluorescence microscope (Olympus, BX53, Tokyo, Japan).
6
Molecular Mechanisms of SDC-1 in MMP-9 Regulation
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Diamidine phenylindole (DAPI) and LPS (Escherichia coli O55:B5) were obtained from Sigma-Aldrich Biotechnology Ltd. Rabbit monoclonal antibodies MMP-9 (ab38898), occludin (ab216327), e-cadherin (ab51034), ZO-1 (ab216880), vimentin (ab92547), α-SMA (ab124964), albumin (ad207327), and active MMP-9 protein (ab168863) were attained from Abcam Biotechnology Ltd. Human and mouse samples of SDC-1 ELISA were bought from Zcibio Biotechnology Ltd (ZC-54317, ZC-37836). Recombinant mouse SDC-1 protein was from RD Biotechnology Ltd. Rabbit monoclonal antibodies GAPDH (ET1601-4), Ly-6G (0809-11), and SDC-1 (ET1703-42) were purchased from Huaan Biotechnology Ltd. HRP-conjugated goat anti-rabbit IgG, Alexa Fluor 488, and 549 goat anti-rabbit IgG were purchased from Bioss Biotechnology Ltd. Sodium cacodylate buffer and lanthanum nitrate were obtained from Zhongjing Instrument Co., Ltd. The human MMP-9 siRNA was constructed by Gene Pharma Biotechnology.
7
Immunohistochemical and Immunofluorescence Analysis of SAT Sections
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Formalin-fixed, paraffin-embedded SAT sections were used for immunohistochemical analysis and immunofluorescence investigations (n = 5 each), as previously described [23 (link)]. For the immunohistochemical analysis, we used anti-GRP78 (Abcam, Cambridge, MA, USA), Imgenex anti-ATF6 (Novus, Littleton, CO, USA), and anti-EPHX2 antibodies (OriGene Technologies, Inc., Rockville, MD, USA). The immunohistochemical analysis results were quantified using ImageScope software version 11.1 (Aperio, Vista, CA, USA), as previously reported [29 (link)]. For immunofluorescent staining, tissue sections were incubated with anti-GRP78 or anti-ATF6 antibody conjugated with Alexa Fluor-488 (Bioss, Woburn, MA, USA). EPHX2 tissue sections were incubated with anti-EPHX2 antibody (OriGene Technologies, Inc., Rockville, USA) and then incubated with Alexa Fluor-488–conjugated goat anti-mouse secondary antibody (Molecular Probes, ThermoFisher, USA). Nuclei were stained using 0.05% DAPI. The sections were viewed with a Zeiss LSM 710 confocal laser-scanning microscope, and representative areas of the sections were photographed under a 40× objective.
8
Immunoblot Antibody Preparation Protocol
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Anti-EIF2A (BS-3613R), anti-IRE1a (BS-8680R), anti-c-Caspase3 (Bioss BS-0081R), anti-Beclin1 (S-1353R) primary antibodies were prepared within 1% BSA at 1/200 dilution, while secondary antibody Bioss Alexa Fluor-488 was prepared at 1/400 dilution manufacturer's instructions.
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