Cocultured THP1 cells were attached to slides with a cytospin, fixed in 4% paraformaldehyde and then rinsed with phosphate-buffered saline. To inhibit nonspecific signals, the slides were exposed to a blocking solution (2% normal goat serum) at room temperature for 2 hours. Then, the slides were incubated with antibodies against CD163 (1:300 dilution; Abcam Ltd., Cambridge, UK) and HLA-DR (an M1 macrophage marker) (1:300, Abcam Ltd.) overnight at 4°C. The slide was rinsed with phosphate-buffered saline three times and then incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibody (1:600; Molecular Probes, Eugene, OR, USA) at room temperature for 2 hours. The samples were rinsed with phosphate-buffered saline three times before they were stained with 4’, 6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA) and examined under a fluorescence microscope (BX50; Olympus, Tokyo, Japan).
Alexa fluor 488 labeled goat anti rabbit igg secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibody is a fluorescent-labeled antibody that binds to rabbit immunoglobulin G (IgG) antibodies. It is designed for use in various immunodetection techniques, such as immunofluorescence microscopy, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp5, by Leica (2 mentions)
Sp5 2, by Leica (2 mentions)
Triton x 100, by CWBIO (2 mentions)
Cofilin, by Santa Cruz Biotechnology (2 mentions)
Slowfade gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (2 mentions)
Anti cortactin mab 4f11, by Merck Group (2 mentions)
Alexa fluor 568 labeled goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (2 mentions)
Anti phosphorylated akt ps473, by Cell Signaling Technology (2 mentions)
Sc 8350, by Santa Cruz Biotechnology (2 mentions)
Nck1 ab23120, by Abcam (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Anti uchl3 rabbit polyclonal antibody, by Bioss Antibodies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lsm800 confocal microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Vectashield antifade mounting media with dapi, by Vector Laboratories (1 mentions)
Perm wash buffer, by BD (1 mentions)
10 protocols using alexa fluor 488 labeled goat anti rabbit igg secondary antibody
1
Immunofluorescence Analysis of THP-1 Macrophage Phenotypes
2
Immunofluorescence Localization of UCHL3 in Spermatozoa
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Smeared semen was fixed in 100% acetone for 15 min at 4°C and stored at –80°C until use. After thawing and warming up to room temperature, the smears were permeabilised with 1% Triton X-100 in phosphate buffered saline (PBS), pH 7.4, for 15 min. After washing three times for 15min with washing buffer [0.2% Tween 20 and 1% inactivated normal goat serum (INGS) in PBS], 5% INGS in PBS was added to block nonspecific sites on the spermatozoa for 50min. Anti-UCHL3 rabbit polyclonal antibody (1:50, Bioss, Beijing, China) was added, and the slides were incubated at 4°C overnight. The antibody diluent was PBS containing 0.5% Tween 20 and 1% INGS. Slides were washed with washing buffer more than three times for 15 min, and then the Alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibody (1:2,000; Molecular Probes, Invitrogen, Carlsbad, CA, USA) was added. Thereafter, slides were washed once with washing buffer (10 min) and twice with PBS (10 min). The nucleus was stained with anti-fade VECTASHIELD® Mounting Medium, including DAPI (H-1200; Vector Laboratories Inc, Burlingame, CA, USA).
The negative control was performed in the same way by substituting non-immune rabbit serum for the primary antibody. Slides were observed under a confocal laser scanning microscope (Leica TCS-SP5, Leica Microsystems, Wetzlar, Germany).
The negative control was performed in the same way by substituting non-immune rabbit serum for the primary antibody. Slides were observed under a confocal laser scanning microscope (Leica TCS-SP5, Leica Microsystems, Wetzlar, Germany).
3
Immunofluorescence Staining of Adherent Cells
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Adherent cells in 6‐well plates culture slides were fixed with 4% paraformaldehyde for 15 minutes; 0.5% Triton X‐100 (CWBIO, China) was used to permeabilize the cells, which were blocked with 3% BSA for 2 hours. Cells were incubated with primary antibody in 3% BSA at 4°C overnight, washed in PBS three times and incubated with Alexa Fluor 488‐labeled goat anti–rabbit IgG secondary antibody (Invitrogen) for 1 hour; nuclei were stained with DAPI solution and then captured by fluorescence microscope (Leica SP5II).
4
Immunofluorescence Analysis of Cortactin and Kinases
5
Immunofluorescence Analysis of Cortactin and Kinases
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Cells were seeded in 5% DMEM-F12 medium with 5% horse serum overnight and treated with 0.2 ng ml−1 EGF for three hours prior to fixation with 4% paraformaldehyde. Fixed cells were permeabilized with 0.05% Triton X-100 and blocked with 3% bovine serum albumin in phosphate-buffered saline. Cortactin was visualized with by immunofluorescence (IF) with anti-cortactin mAb 4F11 (05-180; 1:500) from Millipore and Alexa Fluor 568 labeled goat anti-mouse IgG secondary antibody (A11031; 1:1,000) from Invitrogen. IF assays were also performed with rabbit monoclonal anti phosphorylated AKT (pS473) (4058: 1:200) antibody from Cell Signaling Technology and rabbit polyclonal antibodies against WASP (SC-8350: 1:250) from Santa Cruz, NCK1 (ab23120: 1:200) from Abcam and cofilin (SC-33779:1:200) from Santa Cruz followed Alexa fluor 488-labeled goat anti rabbit IgG secondary antibody (A11034, 1:1,000) from Invitrogen. The nuclei were stained with SlowFade Gold Antifade Mountant with DAPI (S36938) from Life Technologies. Immunofluorescence analysis was carried out using a LSM710 confocal laser scanning microscope (Carl Zeiss).
6
Visualizing Intestinal Gcg and TGR5
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Frozen ileal sections (5 μm) were fixed for 10 min in 4% formaldehyde and then blocked with 4% BSA for 1 h, followed by overnight staining with the primary antibodies against Gcg (1:100, Cell Signalling Technology) and TGR5 (1:100, Abcam). Following primary antibody incubation, the samples were incubated overnight with Alexa Fluor 488–labeled goat anti-rabbit IgG secondary antibody (1:250, Invitrogen, CA, USA). Subsequently, nuclear staining was performed using DAPI (Sigma-Aldrich, MO, USA). Stained specimens were observed using an LSM700 confocal microscope (Carl Zeiss, Oberkochen, Germany), and ImageJ was utilized to measure fluorescence intensity and cell count.
7
Immunofluorescence Assay of MTNR1A/1B
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mLTC-1 cells were cultured in a 35 mm dish with cover slips to a monolayer. Then cover slips were fixed in 4% formaldehyde for 30 min and permeabilized in 0.1% Triton X-100 for 15 min at room temperature. After being blocked with 5% BSA in PBS, cover slips were incubated with a rabbit anti-MTNR1A/1B primary antibody (1:200, Bioss) at 4 °C overnight, followed by incubation with an Alexa-Fluor 488 labeled goat anti-Rabbit IgG secondary antibody (1:300; Invitrogen) for 1 h at 37 °C. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, Beyotime, Beijing, China) for 5 min. Images were captured with a digital camera under a Zeiss LSM800 confocal microscope (Carl Zeiss, Germany).
8
Immunofluorescence Assay for Autophagy Markers
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Adherent cells in 6-well plates culture slides xed with 4% paraformaldehyde for 15 min, 0.5% Triton X-100 (CWBIO, China) was used to permeabilize and blocked with 3% BSA for 2 h. Cells were incubated with primary antibody (Beclin-1 and LC3-II) in 3% BSA at 4°C overnight, PBS washed for three times, incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibody (Invitrogen) for 1 h, nuclei were stained with DAPI solution and then captured by uorescence microscope (Leica SP5II).
9
Fluorescent Visualization of Transfected Proteins in COS7 Cells
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COS7 cells were grown on a coverslip and transfected with the wild type or mutant constructs. After 72 hrs of incubation, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized using 0.2% Tween 20 and blocked for 1 hour with 2.5% bovine serum albumin in phosphate buffered saline (PBS). The cells were then incubated with anti-FLAG primary antibody (1:100) for overnight at 4°C, and then with Alexa fluor 488 labeled goat antirabbit IgG secondary antibody (Thermo Fischer Scientific, Carlsbad, CA, USA) at a dilution of 1:250 for 1 hour at room temperature. Following incubation with primary and secondary antibodies, the cells were washed thrice in PBS containing 0.1% Tween 20. After immunostaining, the cover slips were mounted on slides using Vectashield antifade mounting media with DAPI (Vector Laboratories Inc., Burlingame, CA, USA) and visualized under a fluorescence microscope (Carl-Zeiss Meditec AG, Jena, Germany).
10
Visualizing Anti-HER2 Antibody Trafficking
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Anti-HER2 antibodies lysosomal trafficking was performed according to a published method22 (link). Briefly, N87 cells in growth medium were seeded onto 8-well glass chamber slides (Millicell® EZ slides) at a density of 5 × 104 cells per well. After 18 hr incubation, cells were incubated with Dye-anti-HER2 antibodies (10 nmol/L in PBS) for either 30 mins on ice or 16 hr at 37 °C. After washing twice with cold PBS, the cells were exposed to Fixation/Permeabilization solution (BD Biosciences) for 20 mins at 4 °C and washed twice with 1X Perm/Wash buffer (BD Biosciences). The lysosomal compartment was detected by a rabbit anti-human LAMP2 antibody (GeneTex, cat.GTX103214) and then visualized by Alexa Fluor 488 labeled goat anti-rabbit IgG secondary antibody (ThermoFisher). Cells were mounted with VECTASHIELD mounting medium containing DAPI (H-1200, VECTOR) and examined under a confocal microscope (Leica, TCS SP5).
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