Animals were deeply anesthetized with overdose chloral hydrate (80 mg/kg, i.p.), cardiac perfused with ice-cold phosphate-buffered saline (PBS), and followed by 4% formaldehyde solution. The brains were harvested from the skull and then postfixed for at least 24 h. After postfixation, each brain was sliced coronally (30 mm thickness) using a microslicer (DSK-3000, Dosaka, Kyoto, Japan). The Fos-IR staining was performed using a previous protocol with adjustments (Ohno et al., 2009a, 2011). Briefly, brain slices were washed in PBS with 0.3% Triton X-100, incubated for 2 h in 2% normal rabbit serum, and then incubated again in the presence of goat c-Fos antibody and 2% normal rabbit serum (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 18–36 h. The sections were then washed in PBS and incubated with the biotinylated rabbit anti-goat IgG secondary antibody (Vector Laboratories, Burlingame, CA) for another 2 h. After incubating with the secondary antibody, brain sections were then incubated with PBS containing 0.3% hydrogen peroxide for 30 min. At last, the sections were cleaned with PBS and incubated for 2 h using an avidin-biotinylated horseradish peroxidase complex (Vectastain ABC Kit). The diaminobenzidine-nickel staining method was performed for visualization of Fos staining.
Normal rabbit serum
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Normal rabbit serum is a biological material derived from the blood of healthy rabbits. It is a complex mixture of proteins, antibodies, and other components found in the serum fraction of rabbit blood.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Biotinylated goat anti rabbit igg secondary antibody, by Vector Laboratories (1 mentions)
Mab3400, by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
Bioruptor, by Diagenode (1 mentions)
Mmp 9 antibody, by Santa Cruz Biotechnology (1 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase streptavidin complex, by Beyotime (1 mentions)
Alexa fluor 488 conjugate, by Cell Signaling Technology (1 mentions)
Cover glass bottom dishes, by SPL Life Sciences (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
Triton x 100, by Merck Group (1 mentions)
8 protocols using normal rabbit serum
1
Immunohistochemical Analysis of Fos Expression
2
Cell-Surface P-selectin Translocation Assay
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Endothelial P-selectin translocation was measured by cell surface ELISA as described previously [20 (link)]. MLVECs were challenged with CTH in the presence or absence of specific blocking antibody for TLR2 or TLR4. Reactions were stopped by removing stimulation medium and adding 1% paraformaldehyde for 20 min. The MLVECs were incubated with blocking solution (5% BSA) for 15 min, then with an anti-P-selectin antibody (1:100 dilution, 90 min). Peroxidase activity was quantified at 450 nm using a plate reader. The basal level of cell surface P-selectin from control MLVECs was normalized to 100%. Nonspecific binding was assessed by substituting primary antibodies with normal rabbit serum (Santa Cruz, CA, USA).
3
Characterization of HGFs on PLGA Scaffold
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HGFs from the PLGA scaffold were plated on coverslips and incubated for 6 h at 37°C in an atmosphere containing 5% CO2, in order to allow cells to adhere and proliferate. Cells were harvested and fixed with 4% paraformaldehyde for 30 min at room temperature. Peroxidase activity was blocked using 3% hydrogen peroxide for 30 min at 37°C. After blocking in normal rabbit serum (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), the cells were incubated with monoclonal antibodies against vimentin (cat. no. MAB3404; 1:100) and cytokeratin (cat. no. MAB3400; 1:100) (both from Sigma-Aldrich; Merck KGaA) at 4°C overnight. Following three washes with PBS for 5 min at room temperature, sheep anti-rat immunoglobulin G secondary antibody (1:5,000; cat. no. 5647; Abcam, Cambridge, UK) was added and incubated at 37°C for 1 h. Subsequently, the membrane was analyzed using the UltraSensitive™ S-P Immunohistochemistry kit (cat. no. 40398a; Fuzhou Maixin Biotech. Co., Ltd., Fuzhou, China) according to the manufacturer's protocol. Finally, cells were counterstained with 3% hematoxylin for 60 sec at room temperature and examined using a fluorescence microscope (Olympus Corporation, Tokyo, Japan).
4
GATA3 ChIP-qPCR protocol in breast cancer
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GATA3 antibody was generated in rabbits using recombinant 6x histidine tag-fused GATA3 full-length wild-type protein. ChIP was performed as previously described [24 (link)] with the following modifications. T47D or MCF7 cells were cross-linked with 1% formaldehyde in DMEM F12 for 10 min at room temperature, quenched with glycine, and then sonicated using Bioruptor (Diagenode, Liège, Belgium) to generate 200 to 400 bp DNA fragments. Immunoprecipitation was performed with GATA3 serum, and normal rabbit serum (Santa Cruz Biotechnology, Dallas, TX, USA) was used as a control. The efficiency of the reaction was verified using SYBR-green (Bio-Rad) based Real-Time PCR and primers developed by Eeckhoute et al. [14 (link)] for GATA3 binding sites at ESR1 locus. Quantitation of precipitated DNA was done using a standard curve with 10, 1, 0.1, and 0.01% of input DNA.
5
Immunoprecipitation and Western Blotting
6
Immunohistochemical Analysis of Tissue Sections
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The samples were fixed with 10% buffered formalin and embedded in paraffin. Tissue blocks were cut into 6-µm sections. The sections were blocked with normal rabbit serum (Santa Cruz Biotechnology, Inc.)and then incubated with the same monoclonal antibodies as those used for western blotting (1:100 dilution for STAT6 and Bcl-xL) at 4° C overnight. After washing with TBST, the sections were stained with the horseradish peroxidase streptavidin complex (Beyotime Institute of Biotechnology, Inc.). The sections were color-developed with diaminobenzidine (Beyotime Institute of Biotechnology, Inc.). The sections were stained with hematoxylin and observed under a Leica 400 light microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA).
7
Immunofluorescent Staining of NF-κB p65 in RAW264.7 Cells
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Immunofluorescent staining was performed as previously described [34 (link)]. In brief, RAW264.7 cells were treated with 10 μM of oyster β-thymosin for 2 h and induced with LPS for 24 h on bottom dishes. The cells were stained with DAPI solution at 37 °C for 30 min and then fixed with 4% formaldehyde at room temperature for 15 min. Samples were blocked for 1 h in 5% rabbit normal serum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Cells were incubated with 0.1 μg/mL of NF-κB p65 antibody for 2 h in the dark, then incubated with 0.1 μg/mL of Alexa Fluor® 488 Conjugate (Cell Signaling Technology Inc., Danvers, MA, USA) for another 1 h. The stained cells were mounted on the slides with Prolong Gold Antifade® reagent (Invitrogen, Grand Island, NY, USA) and observed in a confocal laser scanning microscope (CarlZeiss LSM 700, Jena, Germany).
8
Adipocyte Differentiation and PPAR-γ Imaging
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3T3-L1 cells were seeded on cover-glass bottom dishes (SPL Lifesciences, Korea) and differentiated with or without treatment of LEAO. After adipocyte differentiation, cells were washed with 1X PBS and stained by 1 μg/ml of DAPI in methanol for 20 min at 37 o C. After incubation, cells were re-washed with PBS and fixed by using 4% formaldehyde for 10 min at room temperature. Cells were blocked by blocking solution with 5% rabbit normal serum (Santa Cruz Biotechnology Inc., USA) containing 0.3% Triton X-100 (Sigma-Aldrich Inc.) in 1× PBS for 1 h in dark conditions. Blocked cells were treated with anti-PPAR γ primary antibody (Cell Signaling Technology Inc., USA) at 4 o C for overnight. After that, cells were rinsed with 1× PBS and incubated with anti-rabbit IgG (H+L), F(ab'2) fragment (Alexa Fluor 488 conjugate) (Cell Signaling Technology Inc.) for 1 h at room temperature in dark conditions. Cells were washed with 1× PBS and mounted by ProLong Gold Anti-fade Reagent (Invitrogen, USA). The stained cells were observed with a Carl Zeiss LSM 710 confocal laser scanning microscope (Carl Zeiss, Germany).
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