A total of 1 × 104 cells were plated in a glass bottom/confocal petri dish (Biosharp, Hefei, China) and cultured overnight. Cells were fixed by 4% paraformaldehyde solution (Beyotime) for 10 min and blocked with immuno-staining blocking buffer (Beyotime) for 60 min at room temperature, then incubated with mouse anti-EGFR (ProteinTech, 66455-1-lg) and rabbit anti-NK1R (Abcam, ab183713) overnight at 4 °C. The cells were then stained with coralite594-conjugated anti-rabbit IgG (ProteinTech, SA0013-4) and coralite488-conjugated anti-mouse IgG (ProteinTech, SA0013-1) for 2 h at room temperature. Nuclei were stained by DAPI (1 μg/ml, Sangon). Fluorescent images were captured by a microscope equipped with a laser-scanning confocal imaging system (Zeiss LSM800, German).
Immunostaining blocking buffer
Manufactured by Beyotime
Sourced in China
Immunostaining blocking buffer is a solution used in the immunostaining process to block non-specific binding of antibodies, reducing background signals and improving the specificity of the target detection.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (4 mentions)
Immunostaining fix solution, by Beyotime (3 mentions)
Immunostaining permeabilization buffer, by Beyotime (3 mentions)
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi, by Beyotime (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Confocal laser scanning microscope, by Leica (2 mentions)
Paraformaldehyde (pfa), by Beyotime (2 mentions)
Paraformaldehyde solution, by Beyotime (1 mentions)
Coralite488 conjugated anti mouse igg, by Proteintech (1 mentions)
Lsm 800, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Sangon (1 mentions)
Ix73 th4 200 system, by Olympus (1 mentions)
Rabbit anti p65 sc 372, by Santa Cruz Biotechnology (1 mentions)
Hb050 inverted microscope system, by Zeiss (1 mentions)
Dilution buffer, by Beyotime (1 mentions)
Alexa fluor 555 secondary antibody, by Beyotime (1 mentions)
Mouse anti oct4, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti nanog, by Cell Signaling Technology (1 mentions)
Rabbit anti klf4, by Abcam (1 mentions)
Rabbit anti klf4, by Boster Bio (1 mentions)
20 protocols using immunostaining blocking buffer
1
Immunofluorescence Staining of EGFR and NK1R
2
Immunofluorescence Staining of Pig CD163
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The cells were cultured in 24-well plates, rinsed twice with phosphate buffer solution (PBS) and fixed with ice-cold Immunostaining Fix Solution (Beyotime Biotechnology, Shanghai, China) for 20 min, followed by washing with PBS two times. Then, the cells were incubated in ice-cold Immunostaining Permeabilization Buffer (Beyotime, China) for 10 min and washed twice with PBS. The cells were then incubated in Immunostaining Blocking Buffer (Beyotime Biotechnology, China) at room temperature for 60 min, followed by washing with PBS three times. Next, the cells were incubated with the following primary antibody, Mouse anti Pig CD163 Monoclonal Antibody (Bio-Rad, Berkeley, CA, USA, MCA2311GA), at 4 °C overnight, and they were washed twice with PBS. Next, the cells were incubated with Goat anti Mouse IgG (H/L) (DyLight®488, Bio-Rad, USA) for 1 h in the dark at room temperature, following by washing with PBS three times. The cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI) in the dark for 5 min and then rinsed three times with PBS. The images were visualized using an Olympus/IX73 TH4-200 system (Olympus, Tokyo, Japan) or Olympus/FV10i-O system (Olympus, Japan). Three independent experiments were performed for immunofluorescence detection.
3
Double Immunostaining of Neurons
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For double-immunostaining, cells in 6-well plates were fixed with 4% formaldehyde for 10 min at room temperature and then washed three times with PBS. The fixed cells were blocked by treatment with immunostaining blocking buffer (Beyotime Institute of Biotechnology) for 1 h at room temperature. Subsequently, the cells were incubated overnight at 4°C with the primary antibodies: Mouse anti-neuronal nuclei (anti-NeuN; MAB377; 1:200 dilution; EMD Millipore, Billerica, MA, USA), rabbit anti-p65 (sc-372) or anti-c-Rel (sc-272) (1:200; Santa Cruz Biotechnology, Inc.). Goat anti-rabbit IgG (A23220) and goat anti-mouse IgG (A23410) (1:200 dilution; both from Abbkine, Inc., Redlands, CA, USA) were used as secondary antibodies (incubation for 1 h at room temperature). After washing, neurons were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature to stain the cell nuclei. Fluorescence microscopy imaging was performed using a ZEISS HB050 inverted microscope system (Zeiss AG, Oberkochen, Germany).
4
Immunofluorescence Analysis of Pluripotency Markers in mESCs
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J1 mESCs were treated with DMSO or 3 μM CHIR for 24 h on gelatin-coated 12-well plates. The medium was discarded, and the cells were washed twice with phosphate-buffered saline before being fixed and permeabilized with immunostaining fix solution (Beyotime, Jiangsu, China) for 10 min. After blocking with immunostaining blocking buffer (Beyotime) for 1 h, the cells were incubated with primary antibody in dilution buffer (Beyotime) overnight at 4°C and then with Alexa Fluor 555-secondary antibody (Beyotime) for 2 h at room temperature in the dark. After each step, the cells were washed thrice with immunolstaining wash buffer for 5 min before the next step. DAPI (4′, 6-diamidino-2-phenylin-dole) staining was performed after secondary antibody incubation for 10 min at room temperature. The primary antibodies and dilutions used were as follows: rabbit anti-Klf4 (Abcam, Cambridge, UK; 1:500), rabbit anti-Klf4 (Boster, Wuhan, China; 1:500), mouse anti-Oct4 (Santa Cruz, CA, USA; 1:500), rabbit anti-Cdx2 (Santa Cruz; 1:500), and rabbit anti-Nanog (Cell Signaling Technology, Danvers, MA; 1:500). All reagents not indicated were purchased from the Beyotime Institute of Biotechnology (Beyotime). Immunofluorescence staining was visualized and imaged by a confocal microscope (Nikon, Tokyo, Japan).
5
Immunostaining for BTG1 and PRMT1 Proteins
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Cells were grown in culture dishes with a bottom well. Monolayers were washed with PBS, fixed with immunostaining fix solution (Beyotime Institute of Biotechnology) for 1 h at routine temperature, washed with Tris-buffered saline and Triton X-100 (2.42 g Tris, 8 g NaCl, 0.1% Triton X-100) three times, and blocked with immunostaining blocking buffer (Beyotime Institute of Biotechnology) for 1 h. The monolayers were incubated overnight with the BTG1 and PRMT1 antibodies at a temperature of 4°C. After washing, cells were incubated with cyanine 3-conjugated anti-rabbit antibody and FITC-conjugated anti-mouse antibody (Beyotime Institute of Biotechnology) for 1 h at a routine temperature. Finally, specimens were examined under a confocal laser scanning microscope (LSM 710; Zeiss, Oberkochen, Germany).
6
Immunostaining of Cardiac Ion Channels
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Cells were fixed in 4% paraformaldehyde (Boster) for 30 min, and washed in PBS (10 min × 3 times). To block non-specific epitopes, cells were incubated with immunostaining blocking buffer (Beyotime Institute of Biotechnology) for 60 min. Then cells were incubated overnight at 4°C with primary antibodies: c-kit (sc-1494, 1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), HCN1 (ab84816, 1:100), HCN2 (ab65704, 1:100), HCN3 (ab84818, 1:100) and HCN4 (ab69054, 1:100) (all from Abcam), followed by the appropriate fluorescence-conjugated secondary antibodies: Alexa Fluor 488 mouse anti-goat IgG (bs-0294M, 1:200; Bioss, Beijing, China), Alexa Fluor 647 goat anti-mouse IgG (P0191, 1:200) and Alexa Fluor 647 goat anti-rabbit IgG (P0180, 1:200) (both from Beyotime Institute of Biotechnology). Next, cells were incubated with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Beyotime Institute of Biotechnology) to label the cell nucleus. Negative control was performed by omitting the primary antibody. All incubation steps were followed by washes with PBS (10 min × 3 times). Cells were visualized and photographed using a confocal laser scanning microscope (Leica, Wetzlar, Germany). The mean fluorescence density was measured by Image-Pro Plus version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
7
Immunofluorescence Staining of Tubulin in HDFs
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HDFs were cultured in 15-mm diameter confocal petri dishes (NEST, China) overnight. After treatments, cells were fixed with 4% paraformaldehyde for 20 min. Then the cells were washed twice with PBS before blocking in immunostaining blocking buffer (Cat# P0102, Beyotime, China) for 1 h. After incubation with rat anti-tubulin antibody (Cat# ab6160, Abcam, United Kingdom) diluted in 1% bovine serum albumin (BSA) in PBS overnight at 4°C, the dishes were washed with PBS for three times and then stained with Alexa Fluor 488 fluorescent secondary antibody (Cat# A32731, Invitrogen, United States) at 37°C for 1 h in the dark. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (Cat# C1005, Beyotime, China), and the plates were sealed with anti-fluorescence quenching solution (Cat# P0126, Beyotime, China) before imaging. The pictures were acquired using a Leica confocal microscope (Leica Microsystems, Wetzlar, Germany).
8
Immunofluorescence Imaging Protocol
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Cells were fixed with 4% paraformaldehyde and blocked with immunostaining Blocking Buffer (Beyotime, China, P0102). Immunostaining was performed using primary antibody and DAPI. After incubation with fluorescent-labeled secondary antibody. Images were acquired using a confocal microscope under the same conditions for each experiment.
9
Immunostaining of LC3 Protein in H9c2 Cells
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The in situ expression of LC3 protein in H9c2 cells was observed with immunostaining. H9c2 cells were extracted from the medium and washed twice with precooled PBS. After fixation in 4% paraformaldehyde for 15 min, cells were washed with 0.2% Triton in PBS three times for 3 min each time and then blocked with immunostaining blocking buffer (Beyotime Biotech, P0102) at room temperature for 1 h. Primary LC3B antibody was added to the section (1:400, Cell Signaling Tech, 2775) at 4 °C overnight and subsequently reacted with FITC-labeled secondary fluorescence antibody of the corresponding species (1 : 50, Beijing Zhongshan Golden Bridge Biotechnology, ZF-0311). DAPI (Beyotime Biotech, C1005) was used to stain nuclei for 3 min. Sections were mounted and observed under laser confocal microscopy (OLYMPUS, FV1000, Tokyo, Japan). Mean LC3 puncta per cell quantified from 20 different fields was measured by manual counting and DAPI-stained nuclei were counted as cells in the same fields. Then, LC3 puncta per cell were calculated by dividing total dot numbers by quantified nuclei per microscopic field.
10
Visualizing p-P65 Translocation in LPS-Activated RAW 264.7 Macrophages
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Raw 264.7 macrophages (5 × 104/well) were seeded in a 3.5 cm glass bottom plate and treated with LPS + FLs for 12 h. Subsequently, cells were fixed with paraformaldehyde solution (4%) at atmospheric temperature for 30 min and then blocked with immunostaining blocking buffer (Beyotime, Shanghai, China) for 45 min. Cells were incubated with the monoclonal antibody p-P65 (1:100, Cell Signaling Technology, Boston, MA, USA) overnight at 4 °C. FITC-goat anti-rabbit IgG (1:1000, Beyotime, Shanghai, China) stained cells for 1 h, avoiding light. 4′,6-Diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) was added to each plate and incubated for 5 min. Finally, cells were observed under a confocal laser scanning microscope (CLSM, LSM880 Airyscan, Zeiss, Jena, Germany) with a 10× objective lens with NA 0.45 and a 63× oil objective lens with NA 1.4.
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