Confocal microscopy was used to evaluate NETs formation in paraffin-embedded uterine tissue samples. From the paraffin-embedded uterine tissue, an 8 μm thick piece was cut and stained. Briefly, before antigen retrieval, paraffin sections were dewaxed and hydrated, heated in a pressure cooker with Tris-EDTA HIER(Heat-Induced Epitope Retrieval)solution for 2 min at 110 °C, pH = 9. Sections were blocked with Odyssey® blocking buffer and stained at room temperature with a 1:200 dilution of rabbit anti-neutrophil elastase (NE) polyclonal antibody (AB68672, Abcam, Cambridge, UK) and 1:200 dilution of mouse anti-histone 1 monoclonal antibody (MAB3864, Millipore, Burlington, MA, USA) for 1.5 h in a humid chamber. After washing, Alexa Fluor 594 goat anti-mouse IgG (H+L) (1:100, SA00013-3, Proteintech, Chicago, IL, USA) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) (1:50, SA00013-3, Proteintech, Chicago, IL, USA) were used as the secondary antibody, incubated for 1 h at room temperature. The final staining was done with Hoechst 33342 (C0030, Solarbio, Beijing, China) for 5 min, protected from light, and confocal images were taken with a Zeiss LSM 800 confocal microscope.
Sa00013 3
Manufactured by Proteintech
Sourced in China, United States
The SA00013-3 is a laboratory equipment item. It is designed for use in research and analysis procedures. The core function of this product is to facilitate specific laboratory processes, but a detailed description cannot be provided while maintaining an unbiased and purely factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Sa00013 2, by Proteintech (16 mentions)
Sa00013 4, by Proteintech (6 mentions)
Sa00013 1, by Proteintech (3 mentions)
Microtome, by Leica (2 mentions)
Ab16667, by Abcam (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Ab27969, by Abcam (1 mentions)
Ab13303, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab32084, by Abcam (1 mentions)
Ab18203, by Abcam (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab150077, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Proteintech (1 mentions)
Alexa fluor 594 conjugated goat anti mouse igg, by Proteintech (1 mentions)
Tcs sp8 x mp, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Df6647, by Affinity Biosciences (1 mentions)
26 protocols using sa00013 3
1
Confocal Microscopy Visualization of NET Formation
2
Immunofluorescence Analysis of GBM
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Specimens (including 5 normal human brain tissue samples and 5 GBM tissue samples) and cells were performed using antibodies against NOX4 (ab13303, 1 : 200, Abcam, USA), TGF-β1 (ab27969, 1 : 100, Abcam, USA), Vimentin (ab92547, 1 : 200, Abcam, USA), and HIF-1α (20960-1-AP, 1 : 50, Proteintech, China). Fluorescent secondary antibodies (anti-rabbit IgG, SA00013-2, 1 : 500 and anti-mouse IgG, SA00013-3, 1 : 500, Proteintech, China) were used to detect the primary antibodies.
3
Immunofluorescence Analysis of Bioprinted Constructs
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Bioprinted constructs were embedded in Optimal Cutting Temperature (O.C.T.) Compound (Sakura) and frozen at −30 °C. 7 μm sections were obtained by freezing microtome (Leica) and fixed by 10% formalin for 30 min. Specific markers were used to stain sections according to standard immunofluorescence protocols. Briefly, sections were incubated overnight at 4 °C with primary antibodies [rabbit monoclonal anti-beta actin (1:300, ab8227, Abcam), anti-paxillin (1:250, ab32084, Abcam), Ki67 (1:250, ab16667, Abcam), N-cadherin (1:300, ab18203, Abcam)] after antigen retrieval, and blocking. Sections were immersed with the goat anti-rabbit secondary antibody Alexa Fluor® 488 (1:300, ab150077, Abcam) and CoraLite594 goat anti-mouse IgG (1:300, SA00013-3, proteintech) for 2 h in the dark at room temperature. Finally, incubated sections were mounted in DAPI Fluoromount-G (0100-20, Southern Biotech) and pictures were taken with a fluorescence microscope (Olympus, BX51) within 24 h. Mean fluorescence intensity was measured from three random figures in one group by mean grey value from ImageJ.
4
Immunostaining of BV2 Cells for TNF-α and IL-1β
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After various treatments, BV2 cells were fixed in 4% paraformaldehyde and then permeabilized with 0.3% Triton X-100. The cells were blocked with 5% bovine serum albumin (BSA). Subsequently, the cells were immunostained with primary antibodies, including TNF-α (Proteintech, 60291-1-Ig, mouse, 1:50) and IL-1β (Proteintech, 16806-1-AP, rabbit, 1:50). After incubation at 4 °C overnight, the cells were further incubated with the corresponding Alexa fluor 594-conjugated goat anti-mouse IgG (Proteintech, SA00013-3, 1:500) or Alexa fluor 488-conjugated goat anti-rabbit IgG (Proteintech, SA00013-2, 1:500) for 1 h at room temperature. Finally, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma). All images were captured using a confocal microscope (TCS SP8 X & MP, Leica, Germany).
5
Immunofluorescence Localization of HDAC4
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Immunofluorescence assay (IFA) was performed to identify the localization of HDAC4 (stained with rabbit anti-HDAC4 antibody (66838-1-Ig, Proteintech),and tetramethyl rhodamine isocyanate (TRITC)-conjugated goat anti-mouse secondary antibody(SA00013-3, Proteintech)), and 1 µg/ml Dox was added to overexpress Tas fusion and EGFP. HeLa-3 ˟Flag-Tas cells (3 ˟104) were fixed with 4% paraformaldehyde at 25°C for 10 min, which was permeabilized with 0.1% Triton X-100 afterwhile in PBS at 25°C for 10 min. Furthermore, cells were blocked with 3% bovine serum albumin (BSA) in PBS and incubated with anti-Flag antibodies at 25°C for 2 hours. After incubating with rhodamine-conjugated secondary antibodies at 25°C for 45 min, the cells were fixed with 90% glycerol-PBS and examined with an Axio Imager Upright Microscope. Nuclei were stained with DAPI.
6
Immunofluorescent Analysis of Lung Cell Markers
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Sections were incubated with primary antibodies against AQP-5 (Affinity, AF5169, 1:200), SP-C (Affinity, DF6647, 1:200), LC3 (CST, 12741, 1:200) or cleaved caspase-3 (CST, 9661, 1:200) for 60 min, and therewith incubated with secondary coralite488-conjugated anti-rabbit IgG (Proteintech, SA00013-2, 1:10000) or coralite594-conjugated anti-mouse IgG antibodies (Proteintech, SA00013-3, 1:10000) for 60 min. The cells were counterstained with DAPI (Servicrbio, G1012, 1 μg/mL), and observed under a fluorescence microscope (Leica Microsystems).
7
Immunofluorescence Analysis of NR1D1 and LC3
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NIH3T3 cells were subjected to fixation with 4% formaldehyde at 37 °C for 20 min. Subsequently, the cells were washed three times for 5 min each with 0.1M PBS (Gibco, Waltham, MA, USA). Following that, NIH3T3 cells were treated with 0.5% Triton X-100 (Solarbio, Beijing, China) for 20 min, blocked with BSA for 30 min, and incubated overnight at 4 °C with primary antibodies NR1D1 (1:100; Proteintech, 14506-1-AP, Wuhan, China) and LC3 (1:100; Cell Signaling Technology, 83506, Danvers, MA, USA). The next day, the cells were washed three times with PBS. Subsequently, NIH3T3 cells were incubated at room temperature for 30 min with fluorescence-conjugated secondary antibodies (1:3000; Proteintech, SA00013-3 and SA00013-2, Wuhan, China). Following another three washes with PBS, the cells were incubated with DAPI (1:4000; cell signaling Technology; 4083, Danvers, MA, USA) in PBS for 5 min, followed by a final wash with PBS. Cell images were acquired using a LSM 710 Zeiss confocal microscope.
8
Immunohistochemical Analysis of GPX4 and FPN1
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The testes were fixed in 4% FPA overnight and sectioned into slices of 2–3 μm. The sections were rinsed three times in PBS, and endogenous peroxidase was blocked using 3% H2O2 at RT for 15 min. Thereafter, sections were blocked using 5% BSA in 0.3% Triton X-100 in PBS for 1 h at RT. Subsequently, we inoculated the sections overnight with anti-GPX4 antibody (Proteintech, Wuhan, China, 14432-1-AP, 1:1,000 dilution) at 4 °C. Then, we rinsed the sections three times in PBS and inoculated them with a secondary antibody for 1 h at RT. Positive immunoreactivity was performed using DAB and assessed by microscopy (Olympus, Tokyo, Japan). Images were analyzed using Image-Pro Plus 6.0 (Media Cybernetics, MD, US).
For fluorescent staining, the sections were blocked with 3% BSA for 30 min and incubated overnight with anti-GPX4 antibody (14432-1-AP, 1:100, Proteintech Group, Inc) and anti-FPN1 antibody (Cincinnati, OH, US, DF13561) at 4 °C. The following morning, the sections were rinsed three times in PBS and incubated with fluorescent secondary antibodies (SA00013-3, Proteintech, Wuhan, China) for 1 h in the dark at RT. Subsequently, the sections were rinsed in PBS, counterstained with DAPI (Wellbio, Guangzhou, China) and mounted using Prolong Gold anti-fade (Servicebio, Wuhan, China). The samples were then evaluated using a microscope (Olympus, Tokyo, Japan).
For fluorescent staining, the sections were blocked with 3% BSA for 30 min and incubated overnight with anti-GPX4 antibody (14432-1-AP, 1:100, Proteintech Group, Inc) and anti-FPN1 antibody (Cincinnati, OH, US, DF13561) at 4 °C. The following morning, the sections were rinsed three times in PBS and incubated with fluorescent secondary antibodies (SA00013-3, Proteintech, Wuhan, China) for 1 h in the dark at RT. Subsequently, the sections were rinsed in PBS, counterstained with DAPI (Wellbio, Guangzhou, China) and mounted using Prolong Gold anti-fade (Servicebio, Wuhan, China). The samples were then evaluated using a microscope (Olympus, Tokyo, Japan).
9
Immunofluorescent Localization of AQP5 and GPER
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Cell slides and submandibular gland tissue samples were fixed with 4% paraformaldehyde for 20 min, permeabilized in 0.2% Triton X-100 for 20 min, and blocked with 1% bovine serum albumin (BSA) for 1 h. Then the cell slides and submandibular gland tissue samples were incubated with AQP5 antibodies (1:200) at 4 °C overnight, followed by incubation with fluorophore-conjugated secondary antibodies (1:400, SA00013-3, Proteintech) for 1 h at room temperature. DAPI staining for 10 min was carried out after secondary antibody incubation.
To verify whether G protein-coupled estrogen receptor (GPER) was involved in the regulation of salivary secretion in vitro, cell slides were performed the same operation as above and then were incubated with GPER antibodies (1:200) at 4 °C overnight, followed by incubation with fluorophore-conjugated secondary antibodies (1:200, SA00013-2, Proteintech) for 1 h at room temperature. DAPI staining for 10 min was carried out after secondary antibody incubation. Staining was detected using fluorescent microscopy (Olympus, Tokyo, Japan).
To verify whether G protein-coupled estrogen receptor (GPER) was involved in the regulation of salivary secretion in vitro, cell slides were performed the same operation as above and then were incubated with GPER antibodies (1:200) at 4 °C overnight, followed by incubation with fluorophore-conjugated secondary antibodies (1:200, SA00013-2, Proteintech) for 1 h at room temperature. DAPI staining for 10 min was carried out after secondary antibody incubation. Staining was detected using fluorescent microscopy (Olympus, Tokyo, Japan).
10
Immunofluorescence Analysis of Cellular Proteins
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For immunofluorescence assays, cells were plated onto slides (YA0352, Solarbio, Beijing, China), cultured for 12 h, fixed with 4% paraformaldehyde for 20 min at 4°C. Cells were incubated for HGS Rabbit Polyclonal antibody (Proteintech, 10390-1-AP) (1:100), Mouse Monoclonal anti-TUBB3 (Proteintech, 66375-1-Ig), VPS28 antibody (Proteintech, 15478-1-AP) and Anti-VEGFA antibody [EP1176Y]-C-terminal (Abcam, Cat#ab52917), followed by CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) secondary antibodies (Proteintech, SA00013-2) (1:250) or CoraLite594 – conjugated Goat Anti-Mouse IgG(H+L) (Proteintech, SA00013-3), respectively. Cells were transfected with Rab5CA (Q79L)-GFP and pCMV-CD63-mCherry, fixed with 4% paraformaldehyde. Samples were examined with ZEISS LSM880 confocal microscope (Zeiss) and images were processed using ZEN software.
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