Briefly, three-micrometer paraffin sections were incubated overnight at 4 °C with a rabbit polyclonal anti-GFAP antibody (1:500, Z0334, DakoCytomation, Glostrup, Denmark). The sections were then washed with 0.1 M phosphate buffered saline (PBS, pH 7.4) and incubated for 1 h with Alexa Flour 488-conjugated goat anti-rabbit IgG (1:200, A-11008, Life Technologies, Victoria, Australia). The sections were washed with PBS, counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 0.5 µg/mL, D9542, Sigma-Aldrich, St Louis, Missouri, USA), and coverslipped with Dako fluorescent mounting medium (S3023, DakoCytomation). For quantitation, six sections at least 60 μm apart were randomly selected from one eye from each animal. In each section, 6 non-overlapping fields spanning the entire retina were captured at x400 magnification using a Nikon DS-Ri2 camera (Nikon Instruments Inc. NY, USA). ImageJ software was used to set a threshold for immunolabeling which was applied to all fields. Data are presented as the percentage of GFAP immunolabeling per field of retina. Investigators were blinded to the experimental groups. Four to six rats per group were evaluated.
Alexa flour 488 conjugated goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States, Australia
Alexa Fluor 488-conjugated goat anti-rabbit IgG is a secondary antibody that binds to rabbit primary antibodies. It is labeled with the Alexa Fluor 488 fluorescent dye, which can be detected using appropriate filters and instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Anti mouse cd68, by Bio-Rad (2 mentions)
Z0334, by Agilent Technologies (1 mentions)
D9542, by Merck Group (1 mentions)
S3023, by Agilent Technologies (1 mentions)
Ds ri2 camera, by Nikon (1 mentions)
Dako fluorescent mounting medium, by Agilent Technologies (1 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 conjugated donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Orca r2, by Hamamatsu Photonics (1 mentions)
Alexa flour 633 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Tetramethylrhodamine isothiocyanate conjugated phalloidin, by Merck Group (1 mentions)
Fsx100, by Olympus (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Cd64 x54 5 7.1, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Mouse anti cd200r, by Bio-Rad (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
15 protocols using alexa flour 488 conjugated goat anti rabbit igg
1
Quantifying GFAP Immunolabeling in Retina
2
Quantifying Müller Cell Injury in Retina
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Injury to Müller cells was evaluated with immunohistochemistry for glial fibrillary acidic protein (GFAP) using an established method [30 (link)]. Briefly, 3 μm paraffin sections were incubated overnight at 4°C with a rabbit polyclonal anti-GFAP antibody (1:500, cat# Z0334, DakoCytomation). The sections were then washed with PBS and incubated for 45 min with Alexa Flour 488-conjugated goat anti-rabbit IgG (1:200, cat# A-11008, Life Technologies, VIC, Australia), washed again and coverslipped. For quantitation, 4 sections at least 60μm apart were randomly selected from each eye. In each section, 12 non-overlapping fields spanning the mid, central and peripheral areas of retina were captured at x400 magnification using a Spot digital camera (SciTech, VIC, Australia). Image J software was used to set a threshold for immunolabeling which was applied to all fields. Data are presented as the percentage of GFAP immunolabelling per total retinal area in the field. Quantitation was performed in all layers of the retina as well as the mid, central and peripheral retina [30 (link)]. Four to 6 rats per group were evaluated.
3
Immunofluorescence Microscopy of RNA-Binding Proteins
4
Immunostaining of Proteins in Cells
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Cells on cover slips were fixed in 4% formaldehyde in phosphate-buffered saline pH 7.4 (PBS) for 20 min at room temperature, washed with PBS, blocked in 2% horse serum, and permeabilized with 0.02% Triton X-100 in PBS for 30 min at room temperature. Cover slips were then incubated for 2 h at room temperature or overnight at 4°C with anti-MVI (Proteus, USA) antibody diluted at 1 : 50 and anti-AKAP9 antibody (Abcam, UK) diluted at 1 : 200 and washed with PBS, followed by cell incubation with 1 μg/mL Alexa Flour 488-conjugated goat anti-rabbit IgG or Alexa Fluor 555-conjugated donkey anti-goat IgG (both from Molecular Probes, Invitrogen, USA). Vectashield mounting medium (Vector Labs, USA) was used to mount the slides. Alexa488-conjugated bungarotoxin (BTX, from Invitrogen, USA) was used to stain acetylcholine-rich clusters. For negative controls, the primary antibodies were omitted. Images were collected as described in [13 (link)]. Unless stated otherwise, the images represent the confocal 0.3-μm sections of the cell center. MetaMorph software (Leica MM AF Basis Offline Version 1.4.0) was used for the quantification studies.
5
Immunofluorescence Imaging of Yeast Nuclei
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Yeast strains were grown to midlog phase (OD600 ∼0.5) in YPD medium at 23°, processed and labeled as in Ho et al. (2000) (link). Briefly, samples were incubated with anti-Nup116-CTD rabbit antibodies (WU600, Iovine et al. 1995 (link)) overnight at 4°. Bound primary antibodies were detected with Alexa Flour 488-conjugated goat anti-rabbit IgG (1:200, Molecular Probes) and samples were stained with 0.1 mg/mL DAPI. Wide-field images were acquired using a microscope (BX50; Olympus) equipped with a motorized stage (Model 999000, Ludl), Olympus 100× NA1.3 UPlanF1 objective, and digital charge coupled device camera (Orca-R2; Hamamatsu). Images were processed with ImageJ (NIH).
6
Immunofluorescence Microscopy of Cellular Proteins
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For immunofluorescence microscopy, cultured cells were washed twice with phosphate buffered saline (PBS), fixed in 4% paraformaldehyde for 10 min at room temperature, and then permeabilized by incubation for 15 min with 0.1% Triton X-100 in PBS. The samples were blocked with 1% bovine serum albumin followed by incubation with rabbit anti-Smad 2/3 or rabbit anti-β-catenin (Abcam, Ltd., Cambridge, UK) primary antibody overnight at 4 °C. One day after incubation, cells were washed with PBS and incubated with Alexa Flour 488-conjugated goat anti-rabbit IgG (invitrogen, Carlsbad, CA) or Alexa Flour 633-conjugated goat anti-rabbit IgG (invitrogen) secondary antibody, respectively, for 60 min at room temperature. The final antibody treatment also contained tetramethylrhodamine isothiocyanate-conjugated phalloidin (Sigma) for actin and DAPI for nucleus staining (both at 1 µg/mL, Sigma). The slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and cells were viewed under a confocal laser-scanning microscope (LSM510, Carl Zeiss MicroImaging, Thornwood, NY).
7
Visualizing IL-1α and Macrophages in Cecal Tissue
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To detect IL-1α expression in the cecum, frozen cecal sections were incubated with a rabbit anti-IL-1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Alexa Flour 488-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) was used for secondary detection. To detect macrophages in the cecum, frozen sections were incubated with mouse anti-CD68 (Serotec, Oxford, UK). Alexa Flour 568-conjugated goat anti-mouse IgG (Invitrogen) was used for secondary detection. Sections were mounted in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Stained sections were examined and imaged using a fluorescence microscope (FSX100; Olympus, Tokyo, Japan).
8
Multiparametric Flow Cytometry Analysis
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Floating cells were prepared for FACS analysis. Cells were incubated for 20 min with antibodies diluted at the optimal concentrations in FACS buffer (PBS, 5% FBS, 5 mM EDTA, and 1% NaN3). LSRFortessa (BD Biosciences) was used for multiparameter analysis of stained cell suspensions followed by analysis with FlowJo software (Tree Star Inc.). Monoclonal antibodies to mouse CD45 (30-F11), Ly6G (1A8-Ly6g), Ly6C (HK1.4), F4/80 (BM8), CD11b (M1/70), CD3 (145-2C11), CD8α (53-6.7), and CD64 (X54-5/7.1, BD Biosciences) were all from eBioscience unless otherwise indicated. Phospho-PDHE1α (NB110-93479; Novus Biologicals) staining was performed after cell fixation and permeabilization followed by secondary Alexa Flour 488-conjugated goat anti-rabbit IgG (Invitrogen) staining.
9
Immunolabeling of Brain Microglia
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Brain sections were permeabilized by 0.3% Triton-100 in PBS, blocked by 10% goat serum in 90% PBS at room temperature for 1 h, and then incubated primary antibody at 4 °C overnight. The primary antibodies were as follows: mouse anti-CD200R (1:50, Bio-Rad), mouse anti-CD68 (1:300, Bio-Rad), and anti-Iba1 (1:300, Wako, Japan). After rinsed three times, sections were incubated with Alexa Flour 488 conjugated goat anti-rabbit IgG (1:500, Invitrogen) and Alexa Flour 633 conjugated goat anti-mouse IgG (1:1000, Invitrogen) at room temperature for 1 h. The fluorescent imaging was collected by an Olympus fluorescence microscope and processed by ImageJ software.
10
Immunofluorescence Analysis of Epithelial-Mesenchymal Transition
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Immunofluorescence staining was performed as previously described [45 (link)]. Briefly, cells seeded on a 4-well chamber slide (Merck Millipore, Darmstadt, Germany) at a density of 1×104 cells/well were transfected with 1 μg of either pCDNA3.1-CPS1-IT1 or pCDNA3.1 plasmid. Forty-eight hours after transfection, the cells were harvested and fixed in ice-cold acetone for 10 min. The slides were washed 3 times with 1× PBS, blocked with 5% goat serum, and incubated with antibodies against Hsp90, vimentin, N-cadherin and E-cadherin (Cell Signaling Technology). Next, the slides were labeled with Alexa Flour 488-conjugated goat anti-rabbit IgG and Alexa Fluor 546-conjugated goat anti-mouse IgG (Invitrogen) and counter-stained with DAPI. The cells were then analyzed using confocal fluorescence microscopy.
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