Cells were grown on 18mm diameter cover glass (Marienfeld). After 48hr incubation, the cells were rinsed twice with 1X PBS and fixed and permeabilized in methanol-acetone mixture (1:1) for 7min at −20°C. Fixed cells were blocked with 5% BSA/PBS-T (PBS, 0.2% Tween-20) for 1hr at room temperature, and then incubated with CSF-1R antibody (1:250 dilution) and TSC-22 antibody (1:500 dilution) for 16hrs at 4°C. The cells were washed three times with 1X PBS for 5min each and incubated with alexa fluor 488 goat anti-rabbit IgG (Green) and alexa fluor 568 goat anti-mouse IgG (Red) antibody (Invitrogen, 250:1 dilution) in darkness for 1hr at room temperature. Finally, the cells were counterstained with 1μg/ml DAPI for 1min at room temperature and mounted on slides. The signals and co-localization were detected using the confocal fluorescence microscopy
Alexa fluor 488 goat anti rabbit igg green
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 Goat Anti-Rabbit IgG (Green) is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassay applications such as immunofluorescence, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
18mm diameter cover glass, by Marienfeld (2 mentions)
Cover glass, by Marienfeld (1 mentions)
Laser confocal microscope, by Zeiss (1 mentions)
Ab93678, by Abcam (1 mentions)
P36931, by Thermo Fisher Scientific (1 mentions)
Ptm 901, by PTM Biolabs (1 mentions)
Dp 70 digital microscope, by Olympus (1 mentions)
H1009, by Merck Group (1 mentions)
Goat serum, by Vector Laboratories (1 mentions)
Alexa fluor 594 red goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Eclipse 90i, by Nikon (1 mentions)
Pressure cooker, by Biocare Medical (1 mentions)
Image pro plus software version 4, by Media Cybernetics (1 mentions)
Af1042, by R&D Systems (1 mentions)
7 protocols using alexa fluor 488 goat anti rabbit igg green
1
Multicolor Immunofluorescence Microscopy
2
Immunofluorescence Analysis of CLU and eIF3f
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Cells were grown on 18mm diameter cover glass (Marienfeld). After 48hr incubation, the cells were rinsed twice with 1X PBS and fixed and permeabilized in methanol-acetone mixture (1:1) for 7min at −20°C. Fixed cells were blocked with 5% BSA/PBS-T (PBS, 0.2% Tween-20) for 1hr at room temperature, and then incubated with CLU polyclonal antibody (1:300 dilution) and eIF3f polyclonal antibody (1:300 dilution) for overnight at 4°C. The cells were washed three times with 1X PBS for 5min each and incubated with alexa fluor 488 goat anti-rabbit IgG (Green) and alexa fluor 568 donkey anti-goat IgG (Red) antibody (Invitrogen, 1000:1 dilution) in darkness for 1hr at room temperature. Finally, the cells were counterstained with 1μg/ml DAPI for 1min at room temperature and mounted on slides. The signals and co-localization were detected using the confocal fluorescene microscopy.
3
Immunofluorescence Imaging of BRD7 and TSC-22
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Cells were grown on 18 mm diameter cover glass (Paul Marienfeld GmbH & Co.
KG, Lauda-Königshofen, Germany) and transiently transfected with the BRD7
or TSC-22 expression vectors. After 48 h incubation, the cells were rinsed twice
with 1X PBS and fixed and permeabilized in methanol-acetone mixture (1:1) for 7
min at −20°C. Fixed cells were blocked with 5% BSA/PBS-T
(PBS, 0.2% Tween-20) for 1 h at room temperature, and then incubated with
BRD7 polyclonal antibody (1:300 dilution) and TSC-22 polyclonal antibody (1:300
dilution) for overnight at 4°C. The cells were washed three times with 1X
PBS for 5 min each and incubated with alexa fluor 488 goat anti-rabbit IgG
(Green) and alexa fluor 568 donkey anti-goat IgG (Red) antibody (Invitrogen,
Carlsbad, CA, USA, 1,000:1 dilution) in darkness for 1 h at room temperature.
Finally, the cells were counterstained with 1 μg/mL
4’,6-diamidino-2-phenylindole (DAPI) for 1 min at room temperature and
mounted on slides. The signals and co-localization were detected using the
confocal fluorescene microscopy.
KG, Lauda-Königshofen, Germany) and transiently transfected with the BRD7
or TSC-22 expression vectors. After 48 h incubation, the cells were rinsed twice
with 1X PBS and fixed and permeabilized in methanol-acetone mixture (1:1) for 7
min at −20°C. Fixed cells were blocked with 5% BSA/PBS-T
(PBS, 0.2% Tween-20) for 1 h at room temperature, and then incubated with
BRD7 polyclonal antibody (1:300 dilution) and TSC-22 polyclonal antibody (1:300
dilution) for overnight at 4°C. The cells were washed three times with 1X
PBS for 5 min each and incubated with alexa fluor 488 goat anti-rabbit IgG
(Green) and alexa fluor 568 donkey anti-goat IgG (Red) antibody (Invitrogen,
Carlsbad, CA, USA, 1,000:1 dilution) in darkness for 1 h at room temperature.
Finally, the cells were counterstained with 1 μg/mL
4’,6-diamidino-2-phenylindole (DAPI) for 1 min at room temperature and
mounted on slides. The signals and co-localization were detected using the
confocal fluorescene microscopy.
4
Immunofluorescence Localization of MTA1 and TJP1
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5 × 104 cells were seeded on cell slides in 24-well plates. When the cells grew to 60–70% confluent, they were fixed with 4% paraformaldehyde for 20 min and incubated with 0.3% Triton X-100 solution for 20 min. Add 200 μL of rabbit anti-MTA1 and mouse anti-TJP1 (diluted 1:100 with 2% BSA) and incubate overnight at 4 °C. Add 200 μL of Alexa Fluor™ 488 Goat Anti-rabbit IgG (green) and Goat Anti-mouse IgG (red) monoclonal secondary antibody (1:500, Invitrogen, USA), incubated at 37 °C for 40 min. Nuclear counterstaining was performed with 200 μL of DIPA (1:1000 dilution with 1 × PBS). Images were acquired with a laser confocal microscope (ZEISS, Germany) after mounting with anti-fluorescence quenching.
5
Immunohistochemistry of Kidney Tissue
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Formalin-fixed, paraffin-embedded 2- to 3-micron sections were obtained from renal biopsies in the archives of the University of Michigan Department of Pathology. Human kidney biopsies were dewaxed, rehydrated, and subjected to heat-induced antigen retrieval by incubating in 0.1 M citrate buffer (pH 6.0, ab93678, Abcam) using the 2100 Antigen Retriever. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide (H1009, Sigma) in Tris-buffered saline (TBS, pH 7.4) for 30 min. Sections were blocked with 10% normal goat serum in 5% bovine serum albumin at room temperature for 2 h. Sections were incubated with primary antibodies, Kmal (1:150, PTM-901, PTM Biolabs) or Sirt5 (1:250, PA5-31029, Invitrogen), diluted in Da Vinci Green (PD900L, Biocare Medical) at 4 ˚C overnight. Antibody bindings were detected by using secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (green, 1:300, A11034) at room temperature for 1 h. The sections were mounted with gold antifade reagent with DAPI (P36931, Invitrogen). An Olympus DP70 Digital Microscope was used to take pictures by ×20 magnification lenses. Fluorescent signal was quantified with ImageJ.
6
Mcl-1 and γH2AX Immunostaining in PC3 Cells
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Adherent and non-adherent PC3 or PC3/shMcl-1 cells were harvested after a 24 h treatment with 1198, BA, 1198 + BA, or control, applied to slides by smearing, air dried, fixed in formalin for 10 min, permeabilized with 0.1% Triton X-100/PBS for 10 min, rinsed with water, and blocked with goat serum (Vector Laboratories) for 30 min. For DIF, we simultaneously immunostained with rabbit polyclonal Mcl-1 (1/150 dilution) and mouse monoclonal γH2AX (1/200 dilution) for 1 h followed by secondary antibodies Alexa Fluor 488 (green) goat anti-rabbit IgG and Alexa Fluor 594 (red) goat anti-mouse IgG (1/500 dilution; Invitrogen) for 1 h. Mounting medium with DAPI was from Vector Laboratories. Color images were acquired using a Nikon Eclipse 90i fluorescence microscope with FITC/Texas Red filters and merged using Adobe Photoshop 7. A similar DIF experiment was done with PC3/shGFP and PC3/shMcl-1 cells treated with Dox for 4 h.
7
Immunofluorescence Imaging of PDGF and PDGFR in 3D Cell Cultures
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IHCs were performed on paraffin sections (4–5 μm) of NMF and BJ3Z cells cultured in 3D colonies as described [30 (link)]. They used antibodies directed against PDGF-A (sc-7958; rabbit polyclonal, Santa Cruz), PDGF-B (sc-7878; rabbit polyclonal, Santa Cruz), PDGFR-α (AF1062; goat polyclonal, R&D systems) and PDGFR-β (AF1042; goat polyclonal, R&D systems). Briefly, sections were deparaffinized and antigen retrieval was performed in a pressure cooker (Bio-care Medical) at 20 psi for 5 min in citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0). Sections were blocked 30 min with 10% normal goat serum and primary antibodies were applied for 1 hr. Fluorescent secondary antibodies were: Alexa fluor 555 (red) donkey anti-goat IgG (1:300) and Alexa fluor 488 (green) goat anti- rabbit IgG (1:400; both Invitrogen). Cell nuclei were counterstained with 4-6-Diamidino-2-phenylindole (DAPI). Fluorescent images were obtained using a Nikon Eclipse E600 fluorescent microscope coupled to a RGB-MSC micro color camera, and Image Pro Plus software version 4.5 (Media Cybernetics, Silver Spring, MD).
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