Placental tissues were fixed in 4% paraformaldehyde and subsequently embedded in paraffin. Serial sections (3 μm) of paraffin-embedded tissues were analyzed by IF as described elsewhere.23 (link) Briefly, tissue slides were deparaffinized in xylene, rehydrated in a serial ethanol gradient, and blocked with 3% H2O2 for 10 min. The slides were then immersed in TE buffer (10 mM Tris and 1.0 mM EDTA, pH 9.0), warmed in a microwave oven at 92°C–98°C for 15 min for antigen retrieval, and cooled to room temperature. The slides were then blocked with 10% goat serum (Boster, China) for 1 h at room temperature, incubated with primary antibodies overnight at 4°C, and then incubated with fluorescence-labeled secondary antibodies (Bioservice, China) at 37°C for 1 h. The nuclei were subsequently counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Boster, China) and mounted with antifade mounting medium (Boster, China). The cells were fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, and blocked with 10% goat serum (Boster, China). After overnight incubation with primary antibodies at 4°C, the cells were incubated with fluorescence-labeled secondary antibodies (Bioservice, China) at 37°C for 1 h. The nuclei were stained with DAPI, and images were captured with an EVOS microscope (Life Technologies, USA) and/or confocal microscope (Zeiss, Germany).
Goat serum
Manufactured by Boster Bio
Sourced in China, United States
Goat serum is a biological fluid obtained from the blood of goats. It contains a variety of proteins, hormones, and other biomolecules that are essential for cell growth and maintenance.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (18 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (7 mentions)
Triton x 100, by Beyotime (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Antifade mounting medium, by Boster Bio (4 mentions)
Ki67 antibody, by Bioss Antibodies (4 mentions)
Polink 1 hrp dab detection system one step polymer detection system, by ZSGB-BIO (4 mentions)
Paraformaldehyde (pfa), by Solarbio (4 mentions)
Triton x 100, by Solarbio (4 mentions)
Freezing microtome, by Leica (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Confocal laser scanning fluorescence microscope, by Zeiss (3 mentions)
Colorimetric tunel apoptosis assay kit, by Beyotime (3 mentions)
Triton x, by Solarbio (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Confocal microscope, by Zeiss (2 mentions)
Confocal laser scanning microscope, by Leica (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (2 mentions)
Rabbit anti iba1, by Fujifilm (2 mentions)
116 protocols using goat serum
1
Immunofluorescence Analysis of Placental Tissues
2
Immunofluorescence Staining of Cells
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After washing with PBS, cells were fixed in 4% formaldehyde for 15 min and permeabilized with 0.3% TritonX-100 for 10 min, cells were blocked with 10% goat serum (BosterBio, USA) for 1 h and then incubated in primary antibody diluted with 2% goat serum at 4 °C overnight. After incubation with secondary antibodies and visualization of nuclei with DAPI (Beyotime, China), images were processed and analyzed using a confocal laser scanning microscope (Leica, Germany).
3
Fixation and Staining of Mouse Brain Sections
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Mice were sacrificed followed by perfused with ice‐cold phosphate buffer saline (PBS), the brains were isolated and post‐fixed in 4% paraformaldehyde (PFA), then transferred brains into 30% sucrose in PBS solution to make them dehydration until they were totally saturated. Put prepared brains in a freezing microtome (Leica, German) to obtain 15 µm sequential coronal sections, subsequently, the sections were moved onto the glass slides. After permeabilized with 0.3% Triton X‐100 in PBS and blocked with 10% goat serum (BOSTER Biological Technology, China) for 30 min at room temperature, the brain sections were collected to incubate with targeted antibodies according to the previously published procedures.[60 (link)
] When the in vitro cultured cells were used for immunofluorescence assays, cells on coverslips were fixed with 4% paraformaldehyde, permeabilized in 0.3% Triton X‐100, and blocked with 10% goat serum. Cells were then incubated with primary antibody of ADAM10 (Abcam) for 4 °C overnight, stained with goat anti‐rabbit secondary antibody Alexa Fluor 488 (Beyotime, China) for 60 min at room temperature. Then, coverslips were mounted with DAPI Fluoromount‐G (Southern Biotech, Birmingham, Alabama, USA). Images were captured by laser scanning confocal microscope (Leica TCS SP8 X, Germany) and quantified by Image‐J software (National Institutes of Health, USA).
] When the in vitro cultured cells were used for immunofluorescence assays, cells on coverslips were fixed with 4% paraformaldehyde, permeabilized in 0.3% Triton X‐100, and blocked with 10% goat serum. Cells were then incubated with primary antibody of ADAM10 (Abcam) for 4 °C overnight, stained with goat anti‐rabbit secondary antibody Alexa Fluor 488 (Beyotime, China) for 60 min at room temperature. Then, coverslips were mounted with DAPI Fluoromount‐G (Southern Biotech, Birmingham, Alabama, USA). Images were captured by laser scanning confocal microscope (Leica TCS SP8 X, Germany) and quantified by Image‐J software (National Institutes of Health, USA).
4
Immunohistochemical Staining of LYRIC, Fibronectin, α-SMA, and E-cadherin
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Immunohistochemical staining was performed as previously described, with a slight modification [29 (link)]. A GTVisionTM III Detection System/Mo & Rb (Gene Tech, Shanghai, China) was used following the manufacturer’s instructions. All antibodies were freshly diluted with 2% goat serum (Boster, Wuhan, China). The LYRIC (Mtdh) antibody (Abcam, Cambridge, Britain) was used at a 1:100 dilution, the fibronectin antibody (BD Biosciences, USA) was used at a 1:200 dilution, the α-SMA antibody (Sigma-Aldrich, Missouri, USA) was used at a 1:250 dilution and the E-cadherin antibody (BD Biosciences, California, USA) was used at a 1:100 dilution. Slides were counterstained with haematoxylin followed by a bluing reagent. Negative controls were treated with PBS instead of primary antibodies.
5
Immunofluorescence Localization of Nrf2 in RAW264.7 Cells
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To determine the location of Nrf2, RAW264.7 cells were washed with 2 times for 5 min and fixed with solutions (methanol : acetone, 1 : 1) for 30 min at room temperature. The cells were washed with 3 times for 15 min, then incubated with PBST (0.5% TRITON, Sangon biotechnology) for 30 min, and followed by incubation with 10% goat serum (Boster biological technology, Wuhan, China) for 30 min at room temperature. The cells were treated with anti-Nrf2 antibody (1 : 100) for a night at 4°C and 37°C for 30 min. After being washed with PBS, the cells were incubated with a secondary Alexa fluor 488-conjugated goat anti-rabbit IgG antibody (1 : 500) at 37°C for 1 h. RAW264.7 cells were washed for 3 times and nucleus of cells were stained with DAPI (Beyotime Institute of Biotechnology, Shanghai, China) and then analyzed by laser scanning confocal microscopy.
6
Quantifying NET Formation in Neutrophils
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Neutrophils (8 × 104) were seeded into black 96‐well plates coated with poly‐l ‐lysine (Sigma‐Aldrich) and allowed to adhere for 30‐60 minutes before stimulation with APS‐IgG (15 µg/mL), HC‐IgG (15 µg/mL) and PMA (50 nmol/L) or left untreated for 2.5 hours. Cells were then fixed in 4% paraformaldehyde without permeabilization and blocked with goat serum (Boster, Wuhan, China) at 37℃ for 1 hour. NETs were detected with a mouse monoclonal primary anti‐MPO antibody (ab25989; Abcam) diluted 1:200 in blocking buffer overnight at 4℃. Next, the plates were incubated with 1:500 rhodamine‐conjugated anti‐mouse IgG (ZsBio, China) for 1 hour at room temperature, followed by DNA counterstaining with 4′,6‐diamidino‐2‐phenylindole (DAPI; Solarbio). Samples were imaged using ImageXpress Micro Confocal with MetaXpress software. NETs were counted and expressed as a percentage of the total number of cells in the fields.
7
Immunofluorescence Staining of ApoE and Nrf2
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For immunofluorescence staining, tissue sections were deparaffinized by xylene, rinsed, and blocked with goat serum (Boster Biological Technology, Pleasanton, California, USA) at 37°C in a humidified atmosphere for 1 h. Then sections were incubated overnight at 4°C with rabbit monoclonal anti‐ApoE (1:400; Abcam, Cambridge, UK) or rabbit polyclonal anti‐Nrf2 (1:400; Abcam). The sections were rinsed with 0.01 M PBS and incubated with goat IgG conjugated to Alexa 492 (1:200; Earthox, Shanghai, China) for 1 h at 37°C in a dark humidified chamber. Finally, nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (Beyotime Bio, Shanghai, China) for 5 min. After a final rinse, coverslips were mounted with an antifade mounting medium (Beyotime Bio), and sections were observed under a fluorescence microscope.
8
Myometrial Cell Signaling Pathway Assay
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Myometrial cells were seeded in 96 well black-walled clear-bottom plates (Costar, US) (4×104/ml density), and incubated overnight at 37°C in air containing 5%CO2. The blank group and model group were added with 100 μL serum-free medium, and the GZFLC group was added with GZFLC (Jiangsu Kanion Pharmaceutical Co., Ltd. Jiangsu, China, No. 20160319) solution prepared with serum-free medium (25 g⋅L−1) for 1 h. The supernatant was discarded, 100 μL serum-free medium was added to the control group, and 100 μL oxytocin (Cayman, 11,799) (final concentration of 28 μM) was added to the model group and GZFLC group for 15 min respectively. The supernatant was discarded, fixed with paraformaldehyde (Amresco, Cat. No. J531) (0.4 g⋅L−1) for 15 min, washed with PBS for 5 min 3 times, and blocked with 5% goat serum (Boster, Cat. No. 10E12B09)/0.3% tritonx-100 for 1 h. Then cells were incubated with primary antibody against p-SAPK/JNK(CST), p-p44/42 MAPK(Erk1/2) (CST), p-p38(CST) (1:150, 1:150, 1:2000) at 4°C overnight. The next day, it was equilibrated to room temperature, followed by 1 h incubation with Alexa Fluor® 488 Conjugate (1:1000, Invitrogen, United States) at room temperature, washed 3 times with PBS, counterstained with DRAQ5® for 5 min, washed once with PBS, and imaged with a high-content instrument (Thermo, ArrayScanⅦ).
9
Immunofluorescence Staining of Microglia
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The tissue sample was fixed with 4% paraformaldehyde overnight (4 °C) and then dehydrated in a 30% sucrose solution for 48 h (4 °C). The tissue sample was then embedded with optimal cutting temperature compound, placed into isopentane precooled to −80 °C for 10 s, and finally stored at −80 °C. The tissue sample was cut into 20-µm slices, incubated with 0.4% Triton X-100 to disrupt the membrane for 30 min, and blocked with goat serum (Boster, Wuhan, China) for 1 h. The sections were incubated at 4 °C overnight with the primary antibodies rabbit anti-Iba-1 (1:500; Wako, Japan). The unbound primary antibody was washed with PBS on the second day, and the tissue sample was incubated with a secondary antibody for 1 h at RT in the dark. Then washed the slides, and made the slides dry. The antifade mounting medium (including DAPI) was dripped onto the slides, and covered with glass for storage. Confocal laser scanning microscopy (Leica, Wetzlar, Germany) was used to capture images. Fluorescence data were analysed using ImageJ.
10
Quantifying C2C12 Myoblast Fusion
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For measuring fusion, C2C12 myoblasts were washed and fixed with 4% paraformaldehyde for 20 min and then permeabilized in 0.1% Triton X-100 for 10 min. After blocking with goat serum (Boster, China), C2C12 myoblasts were incubated with mouse anti-myosin antibody (R&D, MAB4470) at 1:250 dilution overnight at 4 °C and then incubated with Alexa Fluor 594 labeled secondary antibody (Yeasen, 33212ES60) at 1:500 dilution for 1 h at room temperature and stained with Hoechst 33342 (Invitrogen, H21492) for 5 min at room temperature. The fluorescently stained C2C12 myoblasts were captured by Operetta CLS (PerkinElmer, USA) for analysis. The fusion index and the number of multinuclear myotubes were quantitated based on myosin+ cells per field by Image J software referred to the previous study34 (link).
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