Immunocytochemical staining was conducted on cell-bearing coverslips by the method described elsewhere [15 (link)]. The antibodies used were: Cyclin D1 (Bioss Inc., Beijing, China; 1:100), Tg (Bioss. Inc., Beijing, China; 1:200), E-cadherin (Bioss. Inc., Beijing, China; 1:100), RAR-β (Bioss. Inc., Beijing, China; 1:150), and PPAR-β/δ (Bioss. Inc., Beijing, China; 1:200). Cells incubated in 0.2% DMSO-containing medium were used as a control. The color reaction was performed by using 3,3′-diaminobenzidine tetrahydrochloride (DAB) after the binding of the primary antibody (Vector Laboratories, Burlingame, CA, USA). For double immunofluorescent staining (IF), mouse anti-CRABP2 and rabbit anti-FABP5 were employed in the working concentrations of 1:120. Briefly, the cell-bearing coverslips were washed with phosphate-buffered solution (PBS, pH 7.4), incubated in 3% H2O2 for 10 min and then with anti-CRABP2 (1:120; Proteintech, Chicago, IL, USA) and anti-FABP5 (1:120; Proteintech, Chicago, IL, USA) at 4 °C for one night in a humid chamber. Finally, the coverslips were co-incubated with FITC-conjugated goat anti-mouse IgG and PE-conjugated goat anti-rabbit IgG (both 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37 °C for 60 min in the dark, sealed with fluorescence mounting medium, and observed and imaged under a fluorescence microscope (BX53F, Olympus, Tokyo, Japan).
Pe conjugated goat anti rabbit igg
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The PE-conjugated goat anti-rabbit IgG is a secondary antibody that binds to rabbit primary antibodies. The conjugation with the fluorescent dye Phycoerythrin (PE) allows for the detection and visualization of the target proteins in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Bx53f, by Olympus (2 mentions)
Fitc conjugated goat anti mouse igg, by Santa Cruz Biotechnology (2 mentions)
Lsm 700 system, by Zeiss (2 mentions)
Cyclin d1, by Bioss Antibodies (1 mentions)
E cadherin, by Bioss Antibodies (1 mentions)
Rar β, by Bioss Antibodies (1 mentions)
Ppar β δ, by Bioss Antibodies (1 mentions)
Anti crabp2, by Proteintech (1 mentions)
Anti fabp5, by Proteintech (1 mentions)
Anti α sma, by Thermo Fisher Scientific (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Thermo Fisher Scientific (1 mentions)
Pe conjugated goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Ab51745, by Abcam (1 mentions)
Rabbit anti beclin 1 antibody, by Abcam (1 mentions)
Fitc conjugated donkey anti goat igg, by Santa Cruz Biotechnology (1 mentions)
Anti cd68, by Santa Cruz Biotechnology (1 mentions)
Arginase 1, by BD (1 mentions)
Penicillin and streptomycin, by MP Biomedicals (1 mentions)
8 protocols using pe conjugated goat anti rabbit igg
1
Immunocytochemical Staining of Cell Markers
2
Immunohistochemical Analysis of Apoptosis
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Sections were stained with anti-BAX (Cell Signaling, Danvers, MA, USA), anti-cleaved caspase-3 (ABCAM), anti-α-SMA (ABCAM), or CD68 (Thermo Scientific) at room temperature for 2 hours. TUNEL solution and secondary antibodies (PE-conjugated goat anti-rabbit IgG and PE-conjugated goat anti-mouse IgG [Santa Cruz Biotechnologies]) were then added and sections incubated for 1 hour at 37°C in the dark. TUNEL assays were performed using the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Sections were mounted using Fluoroshield with DAPI and confocal microscopy was performed with a LSM 700 system (Carl Zeiss).
3
Quantifying VEGFA Expression in AN3CA Cells
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AN3CA cells (1×104/group) transfected with vector for 96 h were fixed and stained with anti-VEGFA antibody (1:400, ab51745; Abcam, Cambridge, MA, USA). After three washes with PBS, cells were incubated with PE-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Nuclei were visualized viaDAPI staining and cells were observed under a fluorescence microscope.
4
Dual Immunofluorescence for LC3 and Beclin1
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LC3 and Beclin1 double immunofluorescence labeling was performed on tumor tissues obtained from the two experimental groups. The tumor tissues were rinsed with PBS, and then fixed in cold acetone for 20 min and stored at −20°C. After being blocked with 10% goat serum in PBS for 20 min, tumor tissues were incubated overnight at 4°C with mouse anti-LC3 antibody (1:80; GeneTex Inc., Irvine, CA, USA) and rabbit anti-Beclin1 antibody (1:100; Abcam, Cambridge, UK), followed by co-incubation with FITC-conjugated goat anti-mouse IgG and PE-conjugated goat anti-rabbit IgG (both 1:100; Santa Cruz Biotechnology) at 37°C for 60 min in the dark. Nuclei were labeled with 4,6-diamidino-2-phenylindole, 2-(4-amidinophenyl)-1H-indole-6-carboxamidine. After being sealed with fluorescence mounting medium, the tumor tissues were observed and imaged under a fluorescence microscope (BX53F, Olympus, Tokyo, Japan). The same anti-LC3 and anti-Beclin1 antibodies were used for western blotting at dilutions of 1:200 and 1:600, respectively.
5
Quantitative Analysis of M1 and M2 Macrophages
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Vessel tissue sections were micro cut at room temperature and then deparaffinized through the dewatering process. Subsequently, the sections were triple immunostained with anti-CD68 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), inducible nitric oxide synthase (iNOS, Santa Cruz Biotechnologies) for M1 macrophages, and arginase-1 (BD Biosciences, Franklin Lakes, NJ, USA) for M2 macrophages at 4°C overnight. The sections were washed for 10 min in 1% phosphate-buffered saline (PBS), and then secondarily labeled with FITC-conjugated donkey anti-goat IgG, PE-conjugated goat anti-rabbit IgG, and TR-conjugated goat anti-mouse IgG (Santa Cruz Biotechnologies), respectively, for 1 hour in the dark at room temperature. The sections were washed in PBS for 10 minutes, mounted with Fluoroshield with 4′,6-diamidino-2-phenylindole (DAPI, ImmunoBioscience, Mukilteo, WA, USA), and stored in the dark at 4°C until confocal microscopy was performed with an LSM 700 system (Carl Zeiss, Germany). The quantification of CD68, iNOS and Arg1 were analyzed ZEN software. Then we calculated the co-stained area of each iNOS, Arg-1 in CD68 expression within three representative region of interest rectangles per staining slide (n = 4 vessels/group).
6
Sialidase-mediated Neuraminidase Assay
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The following products were purchased from the vendors indicated: sialidase from Arthrobacter ureafaciens (AUSA, recombinant expressed in Escherichia. coli, Calbiochem, San Diego, CA, USA), X-Neu5Ac (Peptide Institute, Osaka, Japan), halothane (Takeda Pharmaceutical Company, Osaka, Japan), Dulbecco's modified Eagle's medium (DMEM, Wako Pure Chemical Industries, Osaka, Japan), penicillin and streptomycin (MP Biomedicals, Santa Ana, CA, USA), fetal bovine serum (FBS, AusGeneX, Molendinar, QLD, Australia), phycoerythrin (PE)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-CD71 (transferrin receptor) polyclonal antibody (Assay Biotechnology, Sunnyvale, CA, USA). Reagents used for making buffer solutions were purchased from Wako Pure Chemical Industries.
7
Immunohistochemical Analysis of Macrophage Subsets
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Paraffin adipose tissue sections were cut at room temperature and then deparaffinized through the dewatering process. Subsequently, the sections were immunostained with an antibody against macrophage markers F4/80 (Abcam, Cambridge, MA, USA), CD80 (Abcam, Cambridge, MA, USA) for M1 macrophages, and CD163 (Abcam, Cambridge, MA, USA) for M2 macrophages at 4°C overnight. The sections were then washed for 10 min in 1% phosphate-buffered saline (PBS), and incubated at room temperature for 1 hour with biotinylated secondary antibody, PE-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnologies) followed by the Vectastain Elite ABC kit (Vector Labs). A DAB Peroxidase Substrate Kit (Vector Labs) was used to visualize peroxidase reaction. The number of CD163+, CD80+ and F4/80+macrophages was quantified microscopically for each slide from 5-10 randomly chosen fields of five independent mice, as previously described [84 (link)]. All images were captured with a microscope (BX51, OLYMPUS, JAPAN) and analyzed by a blinded observer with ImageJ. Cell numbers were calculated from three randomly-selected microscopic fields, and three consecutive sections were analyzed for each mouse.
8
Yeast Display Library Enrichment
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After three rounds of MACS, the enriched SNU475 yeast display library was induced for FACS. From the first to the third round of FACS, 0.2 × 10 7 -0.8 × 10 7 yeast cells were sorted through FACSCalibur equipped with the sorting accessories (BD Bioscience, San Jose, CA, USA). In the fourth round of FACS, 3.1 × 10 7 yeast cells were run through FACS AriaIII (BD Bioscience).
The induced yeast cells in 100 µl of PBSF (0.8% NaCl, 0.02% KCl, 0.144% Na 2 HPO 4 , 0.024% KH 2 PO 4 , 0.1% BSA, and 0.0744% EDTA-2Na) were labeled with 1 µg of mouse anti-Xpress (Invitrogen) and 5 µg of polyclonal rabbit anti-actin (Invitrogen) antibodies. Two-color labeling was performed with 1 µg of FITC-conjugated donkey anti-mouse IgG (Jackson Labs, PA, USA) and 1 µg of PEconjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, CA, USA). Samples thus prepared could be used for FACS and IF. For IF, an aliquot of the stained yeast cells was loaded onto a glass slide with a cover-glass. The sorted yeast cells were grown in SD-CAA medium at 30°C for 3 days.
The induced yeast cells in 100 µl of PBSF (0.8% NaCl, 0.02% KCl, 0.144% Na 2 HPO 4 , 0.024% KH 2 PO 4 , 0.1% BSA, and 0.0744% EDTA-2Na) were labeled with 1 µg of mouse anti-Xpress (Invitrogen) and 5 µg of polyclonal rabbit anti-actin (Invitrogen) antibodies. Two-color labeling was performed with 1 µg of FITC-conjugated donkey anti-mouse IgG (Jackson Labs, PA, USA) and 1 µg of PEconjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, CA, USA). Samples thus prepared could be used for FACS and IF. For IF, an aliquot of the stained yeast cells was loaded onto a glass slide with a cover-glass. The sorted yeast cells were grown in SD-CAA medium at 30°C for 3 days.
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